Novel cancer antigen eef2

ABSTRACT

The present invention provides a method for detecting cancer using a protein expressed in various cancers, and a pharmaceutical composition for the treatment or prevention of such cancer using the protein as an indicator. Furthermore, the present invention provides a pharmaceutical composition containing a cancer antigen peptide derived from the protein. More particularly, the method comprises the step of determining the presence or amount of an eEF2 polypeptide or an eEF2 antibody in a sample obtained from a subject.

This application is a division of U.S. application Ser. No. 13/143,492,filing date of Sep. 29, 2011, which is the National Stage ofPCT/JP2010/050174, filed Jan. 8, 2010, and claims priority to JP2009-002608 filed Jan. 8, 2009, all of which are incorporated herein byreference.

TECHNICAL FIELD

The present invention provides a method for detecting cancer and apharmaceutical composition for the treatment and prevention of suchcancer. Furthermore, the present invention provides a peptide containingcontiguous amino acids derived from an eEF2 protein having a bindingability to an HLA molecule, and a pharmaceutical composition for thetreatment and prevention of cancer, which contains such a peptide,particularly, an HLA-A*2402-restricted eEF2 peptide, anHLA-A*0201-restricted eEF2 peptide, or an HLA-A*0206-restricted eEF2peptide, and a pharmaceutical composition for the treatment andprevention of cancer, which contains such a peptide, and others. Thepresent application claims priority to Japanese Patent Application No.2009-002608, the whole disclosure of which is incorporated herein byreference.

BACKGROUND ART

Various cancer markers have hitherto been known. However, there are fewcancer markers which can diagnose various cancers using one marker, andsuch cancer markers are intently searched. On the other hand,molecularly-targeted drugs against cancer such as trastuzumab targetingat HER2, imatinib targeting at one of tyrosine kinases, gefitinibtargeting at EGFR, and rituximab targeting at a CD20 antigen are nowdeveloped continuously, but even now there is no pharmaceuticalcomposition for the treatment and prevention of cancer, which targets ateEF2 known as a translation elongation factor (eukaryotic translationelongation factor 2) (Non-Patent Document 1). Also, search of antigenicproteins is carried out with respect to various cancers, but only a fewproteins are proved to be a cancer antigen.

-   Non-Patent Document 1: Nygard 0, Nilsson L., “Kinetic determination    of the effects of ADP-ribosylation on the interaction of eukaryotic    elongation factor 2 with ribosomes”, J Biol Chem. 1990; 265:6030-4

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Thus, an object to be achieved by the present invention is to provide amethod for detecting cancer using a protein expressed in various cancersas an indicator, and a pharmaceutical composition for the treatment orprevention of such cancer detected by the method. Another object of thepresent invention is to provide a pharmaceutical composition containinga cancer antigen peptide derived from such a protein.

Means for Solving the Problems

The present inventors have devoted themselves to much research so as toachieve the above objects. As a result, the present inventors have foundone marker protein eEF2 which is highly expressed in various cancertissues, and accomplished a method for detecting cancer using, as anindicator, expression of the marker protein and an antibody produced inthe body against the marker protein and a pharmaceutical composition forthe treatment and prevention of such cancer which targets at the eEF2protein. Also, the present inventors have found that a part of acontiguous amino acid sequence encoding the eEF2 protein functions as acancer antigen peptide, and proved that such a part can be used in apharmaceutical composition for the treatment and prevention of suchcancer.

Thus, the present invention provides:

(1) A method for detecting cancer in a subject, which comprises the stepof determining the presence or amount of an eEF2 polypeptide, an eEF2antibody or a transcript of an eEF2 gene in a sample obtained from thesubject;(2) The method according to (1), wherein the cancer is selected from thegroup consisting of lung adenocarcinoma, non-small cell lung cancer,small-cell lung cancer, head-and-neck squamous cell cancer, esophagealcancer, esophageal squamous cell cancer, stomach cancer, colon cancer,pancreatic duct cancer, glioblastoma, and malignant lymphoma;(3) A double-stranded siRNA inhibiting cancer cell proliferation,wherein the sense strand consists of the RNA sequence shown in SEQ IDNO:2 and the antisense strand consists of the RNA sequence shown in SEQID NO:3;(4) The double-stranded siRNA according to (3), wherein the cancer cellis derived from cancer selected from the group consisting of stomachcancer, lung cancer, pancreatic cancer, glioblastoma and malignantlymphoma;(5) A pharmaceutical composition for the treatment or prevention ofcancer, comprising the double-stranded siRNA according to (3) or (4) asan active ingredient;(6) A method for the treatment or prevention of cancer, which comprisesadministering an effective amount of the pharmaceutical compositionaccording to (5) to a subject;(7) Use of the double-stranded siRNA according to (3) or (4) for theproduction of a pharmaceutical for the treatment or prevention ofcancer;(8) An shRNA inhibiting cancer cell proliferation, which targets at anmRNA transcribed from the DNA sequence shown in SEQ ID NO:18 or 19;(9) A nucleic acid from which the shRNA according to (8) is transcribed,which has the DNA sequence shown in SEQ ID NO:20 or 22;(10) A vector comprising the nucleic acid according to (9);(11) A pharmaceutical composition for the treatment or prevention ofcancer, which comprises the shRNA according to (8), the nucleic acidaccording to (9) or the vector according to (10);(12) A method for the treatment or prevention of cancer, which comprisesadministering an effective amount of the pharmaceutical compositionaccording to (11) to a subject;(13) Use of the shRNA according to (8), the nucleic acid according to(9) or the vector according to (10) for the production of apharmaceutical for the treatment or prevention of cancer;(14) A pharmaceutical composition for the treatment or prevention ofcancer in an HLA-A*2402-positive subject, comprising an eEF2 peptidehaving an amino acid sequence composed of contiguous amino acids derivedfrom an eEF2 protein, wherein the amino acid sequence is selected fromthe group consisting of:

(a) Arg Phe Tyr Ala Phe Gly Arg Val Phe (SEQ ID NO:4); (b) Ala Phe GlyArg Val Phe Ser Gly Leu (SEQ ID NO:5); (c) Arg Phe Asp Val His Asp ValThr Leu (SEQ ID NO:6); (d) Ala Tyr Leu Pro Val Asn Glu Ser Phe (SEQ IDNO:7); and

(e) an amino acid sequence having substitution, deletion or addition ofone or several amino acids in the amino acid sequences as shown in (a)to (d);(15) The pharmaceutical composition according to (14), wherein the aminoacid sequence is Ala Tyr Leu Pro Val Asn Glu Ser Phe (SEQ ID NO:7);(16) A pharmaceutical composition for the treatment or prevention ofcancer in a subject, comprising a polynucleotide encoding the peptideaccording to (14);(17) The pharmaceutical composition according to any one of (14) to(16), wherein the cancer is selected from the group consisting of lungadenocarcinoma, small-cell lung cancer, esophageal cancer, stomachcancer, colon cancer, pancreatic duct cancer, malignant glioblastoma,malignant lymphoma and head-and-neck squamous cell cancer;(18) A method for the treatment or prevention of cancer, which comprisesadministering an effective amount of the pharmaceutical compositionaccording to any one of (14) to (17) to an HLA-A*2402-positive subject;(19) Use of the peptide according to (14) for the production of apharmaceutical for the treatment or prevention of cancer;(20) A pharmaceutical composition for the treatment or prevention ofcancer in an HLA-A*0201-positive subject, comprising an eEF2 peptidehaving an amino acid sequence composed of contiguous amino acids derivedfrom an eEF2 protein, wherein the amino acid sequence is selected fromthe group consisting of:

(a) Arg Leu Met Glu Pro Ile Tyr Leu Val (SEQ ID NO:8); (b) Lys Leu ValGlu Gly Leu Lys Arg Leu (SEQ ID NO:9); (c) Tyr Leu Asn Glu Ile Lys AspSer Val (SEQ ID NO:10); (d) Ile Leu Thr Asp Ile Thr Lys Gly Val (SEQ IDNO:11); (e) Leu Met Met Tyr Ile Ser Lys Met Val (SEQ ID NO:12); (f) LysLeu Pro Arg Thr Phe Cys Gln Leu (SEQ ID NO:13); (g) Leu Ile Leu Asp ProIle Phe Lys Val (SEQ ID NO:14); and

(h) an amino acid sequence having substitution, deletion or addition ofone or several amino acids in the amino acid sequences as shown in (a)to (g);(21) The pharmaceutical composition according to (20), wherein the aminoacid sequence is Arg Leu Met Glu Pro Ile Tyr Leu Val (SEQ ID NO:8) orIle Leu Thr Asp Ile Thr Lys Gly Val (SEQ ID NO:11);(22) The pharmaceutical composition according to (20), wherein the aminoacid sequence has, in the Leu Ile Leu Asp Pro Ile Phe Lys Val (SEQ IDNO:14), a substitution of the amino acid Ile at position 2 with Leu orMet, and/or a substitution of the amino acid Val at position 9 with Leu;(23) A pharmaceutical composition for the treatment or prevention ofcancer in a subject, comprising a polynucleotide encoding the peptideaccording to (20);(24) The pharmaceutical composition according to any one of (20) to(23), wherein the cancer is selected from the group consisting of lungadenocarcinoma, small-cell lung cancer, esophageal cancer, stomachcancer, colon cancer, pancreatic duct cancer, malignant glioblastoma,malignant lymphoma and head-and-neck squamous cell cancer;(25) A method for the treatment or prevention of cancer, which comprisesadministering an effective amount of the pharmaceutical compositionaccording to any one of (20) to (24) to a subject; and(26) Use of the peptide according to (20) for the production of apharmaceutical for the treatment or prevention of cancer.

Effects of the Invention

According to the present invention, it is possible to detect, in asubject having cancer, or a possibility of cancer, or a prognosis ofcancer, various cancers, for example, lung adenocarcinoma, non-smallcell lung cancer, small-cell lung cancer, head-and-neck squamous cellcancer, esophageal cancer, esophageal squamous cell cancer, stomachcancer, colon cancer, pancreatic duct cancer, glioblastoma, malignantlymphoma and the like in a high sensitivity. Also, it is possible toinhibit proliferation of cancer cells detected by the above method.Furthermore, the present invention provides an HLA-A*2402-restrictedeEF2 peptide or an HLA-A*0201-restricted eEF2 peptide, a pharmaceuticalcomposition for the treatment and prevention of cancer, which comprisessuch a peptide, and others. Accordingly, it is possible to induceeEF2-specific CTL in vivo and in vitro in a subject having HLA-A*2402 orHLA-A*0201. In particular, since about 55% of Japanese have at least oneHLA-A*2402 molecule, and about 19.9% have at least one HLA-A*0201molecule, it is possible to induce the eEF2-specific CTL in a very widerange of subjects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that an eEF2 IgG antibody was detected in sera from lungcancer patients.

FIG. 2 shows the results of immunostaining with an anti-eEF2 antibody inlung tissue sections obtained from patients having non-small cell lungcancer (left) and small-cell lung cancer (right).

FIG. 3 shows the results of immunostaining with an anti-eEF2 antibody intissue sections of head-and-neck squamous epithelium and esophagealsquamous epithelium obtained from patients having head-and-neck squamouscell cancer (left) and esophageal squamous cell cancer (right).

FIG. 4 shows the results of immunostaining with an anti-eEF2 antibody instomach and large colon tissue sections obtained from patients havingstomach cancer (left) and colon cancer (right).

FIG. 5 shows the detection of an eEF2 antibody in sera obtained frompatients having various types of cancers and healthy subjects.

FIG. 6 shows that, in patients having non-small cell lung cancer,subjects indicating a high eEF2 antibody titer in sera have a goodprognosis.

FIG. 7 shows that the double-stranded siRNA targeting at an mRNA of aneEF2 gene inhibits proliferation of stomach cancer cell lines.

FIG. 8 shows that the double-stranded siRNA targeting at an mRNA of aneEF2 gene inhibits cell proliferation of various cancer cell lines.

FIG. 9 shows the cytotoxic activity of CTL induced using an eEF2₇₉₆₋₇₉₄peptide.

FIG. 10 shows the cytotoxic activity of CTL induced using an eEF2₇₉₆₋₇₉₄peptide against endogenous eEF2 gene-expressing cells.

FIG. 11 is a graph showing the results obtained by analyzinginterferon-γ induced using an eEF2₇₃₉₋₇₄₇ peptide by FACS.

FIG. 12 is a graph showing the results obtained by analyzinginterferon-γ induced using an eEF2₆₆₄₋₆₆₉ peptide by FACS.

FIG. 13 is a graph showing that forced expression of an eEF2 proteinaccelerates progression of G2/M phase in a cell cycle.

FIG. 14 is a graph showing that forced expression of an eEF2 proteinaccelerates tumorigenesis in vivo.

FIG. 15 is a graph showing that forced expression of an eEF2 proteinaccelerates tumorigenesis in vivo.

FIG. 16 is a graph showing a cytotoxic activity of CTL induced using aneEF2₇₃₉₋₇₄₇ peptide.

FIG. 17 is a graph showing a cytotoxic activity of CTL induced using aneEF2₅₄₉₋₅₂₇ peptide.

FIG. 18 is a graph showing a cytotoxic activity of CTL induced using aneEF2₆₇₄₋₆₇₉ peptide.

FIG. 19 is a graph showing a cytotoxic activity of CTL induced using aneEF2₆₆₁₋₆₆₉ peptide.

FIG. 20 is a graph showing a cytotoxic activity of CTL induced using aneEF2₃₉₄₋₄₀₂ peptide.

FIG. 21 is a graph showing a cytotoxic activity of CTL induced using aneEF2₂₈₄₋₂₉₂ peptide.

FIG. 22 is a graph showing a cytotoxic activity of CTL induced using aneEF2₂₉₂₋₃₀₀ peptide.

FIG. 23 is a graph showing a cytotoxic activity of CTL induced using aneEF2₂₉₂₋₃₀₀ 2 L peptide.

FIG. 24 is a graph showing a cytotoxic activity of CTL induced using aneEF2₂₉₂₋₃₀₀ 2 M peptide.

FIG. 25 is a graph showing a cytotoxic activity of CTL induced using aneEF2₂₉₂₋₃₀₀ 2L9L peptide.

FIG. 26 is a graph showing the results obtained by measuring theinterferon-γ activity when pulsed with an eEF2₂₉₂₋₃₀₀ peptide ormodified-type eEF2₂₉₂₋₃₀₀ peptides (eEF2₂₉₂₋₃₀₀ 2 L, eEF2₂₉₂₋₃₀₀ 2 M,and eEF2₂₉₂₋₃₀₀ 2L9L peptide).

FIG. 27 is a graph showing inhibition of cancer cell proliferation bynovel shRNAs of eEF2 in vitro. The cell count is shown using percentage(%) of the count of cells into which vectors expressing shRNA of eEF2are introduced, relative to the count of cells into which control vectorshLuc is introduced.

FIG. 28 is a graph showing the results obtained by measuring theinterferon-γ activity when pulsed with a modified-type eEF2₂₉₂₋₃₈₀peptide (eEF2₂₉₂₋₃₀₀ 2 M9L peptide).

FIG. 29 is a graph showing the results of interferon-γ activitymeasurement indicating that seven eEF2 peptides (eEF2₂₉₂₋₃₀₀ peptide,eEF2₇₃₉₋₇₄₇ peptide, eEF2₈₁₉₋₈₂₇ peptide, eEF2₆₇₁₋₆₇₉ peptide,eEF2₆₆₁₋₆₆₉ peptide, eEF2₃₉₄₋₄₀₂ peptide, and eEF2₂₈₄₋₂₉₂ peptide) alsoserve as an HLA-A*0206-restricted peptide.

FIG. 30 is a graph showing the results obtained by measuring theinterferon-γ activity when pulsed with three eEF2 peptides (eEF2₄₀₉₋₄₁₇peptide, eEF2₄₁₂₋₄₂₀ peptide, and eEF2₇₈₁₋₇₈₉ peptide).

BEST MODE FOR CARRYING OUT THE INVENTION

In an aspect, the present invention provides a method for detectingcancer. Subjects in which cancer can be detected using the method of thepresent invention may be any animals such as, for example, human,monkey, mouse, rat, hamster, guinea pig, bovine, horse, sheep, goat,pig, dog, cat, and rabbit, and most preferably human. Although thepresent method can be used even if subject animals are healthy, it ispreferably used in subjects having cancer or a possibility of cancer.Also, the present method can be used in prognosis of cancer treatment insubjects. Characteristics of the present invention reside in the factthat it can detect cancer in early stage as compared with CEA used as aconventional cancer marker. For example, the method of the presentinvention can detect non-small cell lung cancer in early stage,particularly in stage I, in a high sensitivity. In this connection, thestage I refers to a stage representing a tumor state classified into T1or T2, NO and MO in the TNM classification defined by the Union forInternational Cancer Control which is disease stage classification ofmalignant tumors.

The present invention can be practiced using samples obtained from theabove subjects. The samples used in the present invention may be anysamples, and it is possible to use tissues containing cells, forexample. The samples used in the present invention are preferablyvarious types of tissue sections or sera. The samples can be acquiredfrom subjects using techniques conventional to those skilled in the art.In case tissue sections are used as the samples used in the presentinvention, for example, tissues obtained by surgery or biopsy may befixed overnight in 10% formalin, and then embedded in paraffin toprepare thin-sliced sections. On the other hand, in case sera are usedas the samples used in the present invention, peripheral blood ofsubjects may be coagulated in a test tube containing a separating agent,and then, sera may be acquired by centrifugation.

Cancers which can be detected by the method of the present inventioninclude any cancers expressing an eEF2 protein, and are preferably lungadenocarcinoma, non-small cell lung cancer, small-cell lung cancer,head-and-neck squamous cell cancer, esophageal cancer, esophagealsquamous cell cancer, stomach cancer, colon cancer, pancreatic ductcancer, glioblastoma, and malignant lymphoma. In particular, lungadenocarcinoma, small-cell lung cancer, stomach cancer, colon cancer,and malignant lymphoma are preferably detected. Cancers detected in thepresent invention may be those in any stages. For example, cancers inany stage of stage I, stage II and stage III in the TNM classificationdefined by the above International Union Against Cancer may be detected.A cancer which can be detected by the method of the present inventionparticularly early stage is non-small cell lung cancer.

When the detection method of the present invention is practiced in asubject, the presence or amount of an eEF2 polypeptide can be determinedin the above samples. The eEF2 polypeptide in the present inventionmeans a polypeptide having an amino acid sequence of an eEF2 protein ora partial sequence thereof, and includes the following variants. Thus,the eEF2 polypeptide in the present invention may have the amino acidsequence of a human eEF2 protein shown in SEQ ID NO:1; or may have anamino acid sequence having deletion, substitution or addition of one orseveral amino acids in the amino acid sequence shown in SEQ ID NO:1, oran amino acid sequence having deletion, substitution, addition and/orinsertion of one or multiple amino acids in the amino acid sequenceshown in SEQ ID NO:1, for example, an amino acid sequence havingdeletion, substitution or addition of 1 to 9, preferably 1 to 5, 1 to 4,1 to 3, more preferably 1 to 2 amino acids, and still more preferablyone amino acid, or an amino acid sequence having deletion, substitution,addition and/or insertion of 1 to 9, preferably 1 to 5, 1 to 4, 1 to 3,more preferably 1 to 2 amino acids, and still more preferably one aminoacid; or an amino acid sequence having a homology of 70% or more,preferably a homology of 80% or more, more preferably a homology of 90%or more, and still more preferably a homology of 93%, 95%, or 99% ormore as compared with the amino acid sequence shown in SEQ ID NO:1; oran amino acid sequence of a fragment of any one of the above amino acidsequences. The homology of an amino acid sequence can be determinedusing a conventional sequence analyzing tool such as FASTA and BLAST.The fragment in the present invention refers to a portion of the aboveeEF2 polypeptides. Also, the eEF2 polypeptide in the present inventionincludes polypeptides which have properties comparable to those of theeEF2 protein and which have an amino acid sequence encoded by anucleotide sequence hybridizing with a nucleotide sequence encoding theamino acid sequence shown in SEQ ID NO:1 under a stringent condition.The comparable properties in the present specification refer tobiologically, chemically and physically comparable properties ascompared with the eEF2 protein. The eEF2 protein in the presentinvention is derived from human. Even if, however, the eEF2 protein isderived from other animals such as, for example, mouse, monkey, rat,bovine and cat, the eEF2 protein in these animals is included in theeEF2 protein in the present specification.

In the present invention, the conditions of the above hybridization canbe selected suitably by those skilled in the art according to thedescription of J. Sambrook et al., “Molecular Cloning: A LaboratoryManual, Second Edition”, 1989, Cold Spring Harbor Laboratory Press.Although the conditions of the hybridization may be a low stringentcondition, a high stringent condition is preferable. The low stringentcondition is, for example, a condition of 42° C., 0.1×SSC and 0.1% SDS,preferably a condition of 50° C., 0.1×SSC and 0.1% SDS, in a washingstep after hybridization in accord with the above reference. The highstringent condition includes, for example, a condition of 65° C., 5×SSCand 0.1% SDS, etc. However, those skilled in the art can realize similarconditions by suitably selecting the above elements.

The detection method of the present invention may be carried out by anymethods. For example, the detection method of the present invention canbe carried out using an antibody against the above eEF2 polypeptide. Anantibody against a polypeptide having an amino acid sequence in anarbitrary region of the above eEF2 polypeptide may be used in thedetection method of the present invention. For example, an antibodyagainst a polypeptide having a region of positions 1-417 or positions411-858 in the amino acid sequence of the human eEF2 protein may beused. The antibody used in the present invention may be any isotype ofIgG, IgA, IgM, IgD and IgE. Also, the antibody used in the presentinvention may be a monoclonal antibody or a polyclonal antibody. Theantibody used in the present invention may be prepared using aconventional technique, or may be a marketed product.

Also, the detection method of the present invention can be carried outusing an antibody against an eEF2 antibody. The eEF2 antibody which canbe detected in the present invention is one produced in vivo, i.e., inthe body of a subject. In the detection method of the present invention,an antibody against the above eEF2 antibody can be prepared by a knowntechnique, or may be a marketed product. Preferably, an anti-eEF2antibody (H-118, Santa Cruz Biotechnology, Santa Cruz, Calif.) may beused.

In order to determine the presence or amount of an eEF2 polypeptide oran eEF2 antibody in a sample, known means and methods can be used in thepresent invention. Any means and methods may be used so far as they candetect qualitatively or quantitatively an eEF2 polypeptide or an eEF2antibody. For example, they include immunological detection methods fora protein such as immunostaining, dot blotting, fluorescence antibodytechnique, complement-binding reaction, neutralizing antibodymeasurement, immunoprecipitation, western blotting, radioimmunoassay(RIA), ELISA, and two-hybrid system. Preferably, immunostaining or dotblotting may be used in the present invention.

“Positive” evaluation can be determined in the detection method of thepresent invention by comparing the presence or amount of an eEF2polypeptide or an eEF2 antibody in a sample obtained from a subject withthe presence or amount of the eEF2 polypeptide or eEF2 antibody in asample obtained from a healthy subject or a subject in a normal phase.In case a serum is used as a sample in the detection method of thepresent invention, an antibody titer (densitometric units) of an eEF2antibody in the serum may be used as an indicator. In this case, anantibody titer of an eEF2 antibody in a serum of a subject is measuredby dot blotting, and a numerical value higher than that in a serum froma healthy subject, preferably 1,000 or more, more preferably 2,000 ormore of an antibody titer (densitometric units) may be determined as“positive”. However, the numerical value can vary depending on variousfactors such as cancer types and tissues, and can be set suitably bythose skilled in the art. On the other hand, in case a tissue section isused as a sample, a degree of stain by immunostaining in the tissuesection may be used as a criterion. In this case, for example, thepresence of cancer cells showing intense stain as compared withcorresponding normal cells in an amount of 25% or more of total cancercells may be determined as “positive”. However, the determination may bemade suitably by those skilled in the art.

When the detection method of the present invention is practiced in asubject, the presence or amount of a transcript of an eEF2 gene can bedetermined in a sample. The transcript of an eEF2 gene in the presentinvention means a product transcribed from a nucleotide sequenceencoding an amino acid sequence of the above eEF2 polypeptide or afragment thereof, and may be, for example, mRNAs or any other types ofRNAs as well as their fragments, etc. Also, the presence or amount of apolynucleotide having a nucleotide sequence (for example, DNA sequence)encoding an amino acid sequence of an eEF2 polypeptide or a fragmentthereof can be determined in the detection method of the presentinvention.

In order to determine the presence or amount of the above transcript orpolynucleotide in a sample, means and methods conventional to thoseskilled in the art as a method for detecting a polynucleotide may beused in the present invention. For example, they include methods fordetecting a polynucleotide such as in situ hybridization, northernblotting, southern blotting, dot blotting, RNase protection assay, PCR,RT-PCR, and real-time PCR. Also, it may be possible to carry out a geneanalyzing method using a microarray (for example, DNA microarray,microRNA microarray, protein microarray, etc.). Furthermore, othermethods may be used so far as they can detect the above transcript orpolynucleotide qualitatively or quantitatively.

Furthermore, the method of the present invention can be used fordiagnosis of prognosis of a subject having cancer. Cancers to which themethod of the present invention can be applied may be any cancersexpressing an eEF2 protein as described above, and they are preferablylung adenocarcinoma, non-small cell lung cancer, small-cell lung cancer,head-and-neck squamous cell cancer, esophageal cancer, esophagealsquamous cell cancer, stomach cancer, colon cancer, pancreatic ductcancer, glioblastoma, malignant lymphoma, and more preferably non-smallcell cancer. In the diagnosis of prognosis in the present invention, thehigher the value of an antibody titer of an eEF2 antibody in a sampleobtained from a subject, the better the prognosis. For example, theantibody titer (densitometric units) of the eEF2 antibody is a value of1,000 or more, preferably 2,000 or more, and more preferably 4,000 ormore, and those skilled in the art can suitably determine the valuetaking various factors into account.

In another aspect, the present invention relates to a diagnosis kit fordetecting cancer, which comprises, as an essential constituent, anantibody against the above eEF2 polypeptide or eEF2 antibody, or apolynucleotide probe complementary to the above transcript of eEF2 geneor a portion thereof. In the present invention, the above antibody orprobe is preferably labeled. The above labeling can be carried out by aconventional method. The kit of the present invention contains, forexample, a reagent essential to a method for detecting a protein or apolynucleotide, a sampling means, a reaction vessel and the like, inaddition to the antibody against the above eEF polypeptide or eEF2antibody, or the polynucleotide probe complementary to the abovetranscript of eEF2 gene or a portion thereof. In general, the kit isaccompanied with an instruction manual. The kit of the present inventioncan be used to detect efficiently cancer expressing an eEF2 protein in aserum or a tissue.

In another aspect, the present invention relates to a double-strandedsiRNA which inhibits cancer cell proliferation. Cancer cells of whichproliferation can be inhibited by the present invention may be anycancers expressing an eEF2 protein, and are preferably lungadenocarcinoma, non-small cell lung cancer, small-cell lung cancer,head-and-neck squamous cell cancer, esophageal cancer, esophagealsquamous cell cancer, stomach cancer, colon cancer, pancreatic ductcancer, glioblastoma, and malignant lymphoma. In particular, lungadenocarcinoma, small-cell lung cancer, stomach cancer, colon cancer,and malignant lymphoma are preferably inhibited.

The siRNA of the present invention is a double-stranded siRNA containinga sense strand and an antisense strand targeting at a nucleotidesequence of an mRNA transcribed from a human eEF2 gene. The nucleotidesequence targeted by the siRNA of the present invention may be a partialsequence of a nucleotide sequence encoding the amino acid sequence shownin SEQ ID NO:1. The siRNA of the present invention is preferably adouble-stranded siRNA consisting of the sense strand (SEQ ID NO:2) andthe antisense strand (SEQ ID NO:3) having the RNA sequences as shownbelow:

Sense strand of siRNA of the present invention; (SEQ ID NO: 2)5′-CAUGGGCAACAUCAUGAUCGAUCCUGUCCU-3′Antisense strand of siRNA of the present invention; (SEQ ID NO: 3)5′-AGGACAGGAUCGAUCAUGAUGUUGCCCAUG-3′.

Although preferred RNA sequences of the siRNA of the present inventionare the above sequences shown in SEQ ID NOs:2 and 3, these sequences mayhave addition, deletion or substitution of one, two or three bases.Also, these sequences may have substitution, deletion, addition and/orinsertion of 1 to 3, preferably 1 or 2 bases, and more preferably onebase. The hybridization condition in this case is a condition in aliving body in case the siRNA of the present invention is used byadministering in a living body, and a moderately stringent condition ora high stringent condition in case the siRNA of the present invention isused in vitro as a reagent. Such a condition includes, for example, ahybridization condition of 400 mM NaCl, 40 mM PIPES pH6.4, 1 mM EDTA, at50° C. to 70° C. for 12 to 16 hours. Also, the sense strand sequence ofthe siRNA of the present invention has a sequence homology of 90% ormore, preferably 95% or more, and more preferably 95, 96, 97, 98, or 99%or more to a target sequence.

Also, the siRNA of the present invention may have addition of anoverhang sequence at 5′ end or 3′ end. In this connection, the overhangsequence refers to a protruding sequence added to either 5′ end or 3′end of a double-stranded sequence consisting of paired sense andantisense strands in order to increase a stability of thedouble-stranded siRNA. The overhang sequence includes, for example, asequence such as AG, UA, AUG, UUG and AAGCUU from 5′ side, and anysequences can be used. In the double-stranded siRNA of the presentinvention, UU is preferably added to the 3′ end of sense and antisensestrands. Also, the above double-stranded siRNA of the present inventionmay form an shRNA by linking two siRNAs through a loop sequence.

The double-stranded siRNA of the present invention can be prepared by amethod conventional to those skilled in the art. For example, it may besynthesized in vitro chemically or enzymatically, or synthesized invivo, but the method is not limited thereto. A chemically synthesizingmethod is preferably used. After each strand is synthesized by such asynthetic method, the strands can be paired under a conventional pairingcondition. When used, the strands may be purified suitably as needed.Also, the double-stranded siRNA of the present invention may be preparedin the form of a siRNA expression vector expressing the above RNAsequences of the siRNA (SEQ ID NO:2 and SEQ ID NO:3). In this case, forexample, tRNA-shRNA expression vector, piGENE tRNA Pur (Clontech, PaloAlto, Calif.) may be used for the preparation. There is no particularlimitation on the length of the siRNA used in the present invention, and15 to 50 mer siRNA can be exemplified as an example of a preferred siRNAof the present invention, 20 to 40 mer siRNA as a more preferredexample, and 25 to 35 mer (for example, 30 mer) siRNA as furtherpreferred example. Thus, a double-stranded siRNA which can hybridizewith the sequence: 5′-CAUGGGCAACAUCAUGAUCGAUCCUGUCCU-3′ (SEQ ID NO:2) ofthe eEF2 mRNA and which is 15 to 50 mer, preferably 20 to 40 mer, morepreferably 25 to 35 mer (for example, 30 mer) in length of each siRNAcan be exemplified as an example of a preferred siRNA of the presentinvention.

In general, it is known that a siRNA binds to an mRNA of a target genein cells into which the siRNA is introduced and inhibits expression ofthe mRNA. Accordingly, the double-stranded siRNA of the presentinvention has a function of inhibiting expression of an eEF2 gene,thereby being able to inhibit cell proliferation in a subject into whichthe siRNA is introduced. Methods for introducing or administering thesiRNA in the present invention may be those known to those skilled inthe art such as a calcium phosphate method using a transfection reagent,a liposome method, a non-liposome method, electroporation, and amagnetic particle method.

Alternatively, a method may be adopted in which the siRNA is integratedinto a conventional siRNA expression vector and the vector is introducedby a known method as described above. Preferably, a siRNA expressionvector expressing the above RNA sequences of the siRNA (SEQ ID NO:2 andSEQ ID NO:3) is introduced by a known method. Also, the siRNA of thepresent invention may be administered in the form of a pharmaceuticalcomposition as described below.

The present invention provides a pharmaceutical composition for thetreatment or prevention of cancer, comprising the above double-strandedsiRNA as an active ingredient. The pharmaceutical composition of thepresent invention may contain a known anticancer drug as an activeingredient, in addition to the above double-stranded siRNA.

The pharmaceutical composition of the present invention may contain asiRNA as an active ingredient in the form of a vector into which thesiRNA is integrated. For example, the siRNA may be contained in the formof one cloned into a known siRNA expression vector such as acommercially available siRNA expression vector or a known siRNAexpression vector suitably recombined according to an aspect used.Accordingly, the present invention provides a nucleic acid encoding thesiRNA of the present invention, and a vector containing the nucleicacid.

The pharmaceutical composition of the present invention may contain apharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier which can be used in the present invention may be one or morecomponents selected from the group consisting of a physiological saline,distilled water, Ringer's solution, a buffered physiological saline, adextrose solution, a maltodextrose solution, glycerol, ethanol and aliposome, but is not limited thereto. Also, other conventional additivessuch as an antioxidant, a buffered aqueous solution, and abacteriostatic agent may be added to the pharmaceutical composition ofthe present invention. Furthermore, diluents, sprays, surfactants,binders and lubricants may be added to the composition in order toproduce an injection solution, pills, capsules, granules or tablets.

The dosage form of the pharmaceutical composition of the presentinvention may be oral administration or parenteral administration (forexample, intravenous administration, intradermal administration,subcutaneous administration, intramuscular administration, transnasaladministration or oral administration), and other dosage forms may alsobe used so far as they can deliver an active ingredient efficiently toan affected part or its neighborhood. The effective amount of the siRNAof the present invention administered through the pharmaceuticalcomposition of the present invention can be determined depending onconditions of subjects such as, for example, weight, age, sex and healthstate of subjects, as well as amount of food, frequency ofadministration, method of administration, amount of excretion,seriousness of disease and the like. The effective amount of the siRNAof the present invention administered through the pharmaceuticalcomposition of the present invention is usually from 0.01 to 100 mg/kgper day, and preferably from 0.1 to 10 mg/kg per day.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administering aneffective amount of the above pharmaceutical composition to a subject.The cancers to be treated or prevented may be any cancers so far as theyexpress an eEF2 protein, and include, for example, lung adenocarcinoma,non-small cell lung cancer, small-cell lung cancer, head-and-necksquamous cell cancer, esophageal cancer, esophageal squamous cellcancer, stomach cancer, colon cancer, pancreatic duct cancer,glioblastoma, and malignant lymphoma. Preferably, the composition of thepresent invention may be administered to a subject who is determined as“positive” by the above method for detecting cancer.

In still another aspect, the present invention relates to use of theabove double-stranded siRNA for the production of a pharmaceutical forthe treatment or prevention of cancer.

In still another aspect, the present invention relates to an shRNA orsiRNA which inhibits cancer cell proliferation. In general, the shRNA(short hairpin RNA or small hairpin RNA) is an RNA in which a sensestrand and an antisense strand are linked through a loop sequence, andmay produce a double-stranded siRNA by intracellular cleavage of theloop structure. The siRNA of the present invention is preferably adouble-stranded siRNA. There is no particular limitation on regions inthe eEF2 targeted by the shRNA or siRNA of the present invention, and anshRNA or siRNA can be exemplified as a preferred example which targetsat an mRNA transcribed from the following DNA sequence: 5′-gcctggccgagga catcgataaa ggcgagg-3′ (SEQ ID NO:18); or 5′-actcaaccataacactt gatgccgttt ctt-3′ (SEQ ID NO:19). The shRNA of the presentinvention may be one transcribed from a vector containing a DNA sequenceconsisting of sense sequence-loop sequence-antisense sequence of theabove DNA sequence. When transcribed from a vector containing such a DNAsequence to an RNA, RNAs derived from a sense sequence and an antisensesequence bind to each other to form a short hairpin RNA, and arestabilized. In this connection, the loop sequence used in the presentinvention may be any sequences, which can be selected suitably by thoseskilled in the art. An siRNA which can hybridize with an mRNAtranscribed from 5′-gcc tggccgagga catcgataaa ggcgagg-3′ (SEQ ID NO:18)or 5′-actcaac cataacactt gatgccgttt ctt-3′ (SEQ ID NO:19) can beexemplified as a preferred example of the siRNA of the presentinvention. An siRNA which has a sequence complementary to an mRNAtranscribed from 5′-gcc tggccgagga catcgataaa ggcgagg-3′ (SEQ ID NO:18)or 5′-actcaac cataacactt gatgccgttt ctt-3′ (SEQ ID NO:19) can bementioned as a more specific example of the siRNA of the presentinvention. Alternatively, the siRNA of the present invention may be adouble-stranded siRNA composed of a sense strand and an antisense strandof an mRNA corresponding to the DNA sequence shown in SEQ ID NO:18 or19. An shRNA containing an RNA which can hybridize with an mRNAtranscribed from 5′-gcc tggccgagga catcgataaa ggcgagg-3′ (SEQ ID NO:18)and an RNA which can hybridize with the RNA can be exemplified as apreferred example of the shRNA of the present invention. Also, an shRNAcontaining an RNA which can hybridize with an mRNA transcribed from5′-actcaac cataacactt gatgccgttt ctt-3′ (SEQ ID NO:19) and an RNA whichcan hybridize with the RNA can be exemplified as a preferred example ofthe shRNA of the present invention. An shRNA can be mentioned as a morespecific example of the shRNA of the present invention, which istranscribed from a nucleic acid having the following DNA sequence:

(SEQ ID NO: 20) 5′-gcc tggccgagga catcgatgaa agcgtgg cttcctgtcacctcgcc tttatcgatg tcctcggcca ggc-3′; (SEQ ID NO: 22)5′-actcaac cataacactt gataccattt gtt cttcctgtcaaag aaacggcatc aagtgttatg gttgagt-3′; (SEQ ID NO: 21)3′-cgg accggctcct gtagctactt tcgcacc gaaggacagtggagcgg aaatagctac aggagccggt ccg-5′; or (SEQ ID NO: 23)3′-tgagttg gtattgtgaa ctatggtaaa caa gaaggacagtttc tttgccgtag ttcacaatac caactca-5′.In the present invention, the above DNA sequence or RNA sequence mayhave addition, deletion or substitution of 1, 2, or 3 bases.Alternatively, the sequence in the present invention may have a sequencehomology of 90% or more, preferably 95% or more, and more preferably 95,96, 97, 98, or 99% or more to the above DNA sequence or RNA sequence. Inthis connection, the homology, condition of hybridization, and length ofthe siRNA are as described above. Also, in the present invention, theabove DNA sequence or RNA sequence may have addition, deletion,substitution and/or insertion of 1, 2, or 3 bases.

The shRNA or siRNA of the present invention can be prepared by a methodconventional to those skilled in the art. For example, it may besynthesized in vitro chemically or enzymatically, or synthesized invivo, but the method is not limited thereto. Also, the shRNA or siRNA ofthe present invention may be prepared in the form of an shRNA expressionvector or a siRNA expression vector containing a DNA sequence as shownin the above SEQ ID NO:20 or 22. In this case, for example, tRNA-shRNAexpression vector, piGENE tRNA Pur (Clontech, Palo Alto, Calif.) may beused for the preparation.

Methods for introducing or administering the shRNA or siRNA of thepresent invention may be those known to those skilled in the art such asa calcium phosphate method using a transfection reagent, a liposomemethod, a non-liposome method, electroporation, and a magnetic particlemethod. Alternatively, a method may be adopted in which the RNA isintegrated into a conventional siRNA expression vector and the vector isintroduced by a known method as described above. The shRNA or siRNA ofthe present invention may be administered in the form of apharmaceutical composition as described below.

The present invention provides a pharmaceutical composition for thetreatment or prevention of cancer, comprising the above shRNA or siRNAas an active ingredient. The pharmaceutical composition of the presentinvention may also contain a known anticancer drug.

The pharmaceutical composition of the present invention may contain anucleic acid encoding the shRNA or siRNA of the present invention (forexample, a nucleic acid containing a DNA sequence shown in SEQ ID NO:20or 22) as an active ingredient. For example, the nucleic acid may be onein which a nucleic acid containing such a DNA sequence is integratedinto a commercially available shRNA expression vector or siRNAexpression vector. Thus, the present invention provides a nucleic acidencoding the shRNA or siRNA of the present invention, and a vectorcontaining the nucleic acid.

The pharmaceutical composition of the present invention may containpharmaceutically acceptable conventional carriers and additives. Thedosage form of the pharmaceutical composition of the present inventionmay be oral administration or parenteral administration (for example,intravenous administration, intradermal administration, subcutaneousadministration, intramuscular administration, transnasal administrationor oral administration). Also, the effective amount of the shRNA orsiRNA of the present invention administered through the pharmaceuticalcomposition of the present invention can be determined depending onconditions of subjects such as, for example, weight, age, sex and healthstate of subjects, as well as amount of food, frequency ofadministration, method of administration, amount of excretion,seriousness of disease and the like.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administering aneffective amount of the above pharmaceutical composition to a subject.Cancers to be treated or prevented may be any cancers so far as theyexpress an eEF2 protein, and include, for example, lung adenocarcinoma,non-small cell lung cancer, small-cell lung cancer, head-and-necksquamous cell cancer, esophageal cancer, esophageal squamous cellcancer, stomach cancer, colon cancer, pancreatic duct cancer,glioblastoma, and malignant lymphoma.

In still another aspect, the present invention relates to use of theabove shRNA or siRNA for the production of a pharmaceutical for thetreatment or prevention of cancer.

In a further aspect, the present invention relates to a peptidecontaining contiguous amino acids derived from an eEF2 protein. Examplesof the peptide of the present invention are peptides containing an aminoacid sequence as described below or peptides consisting of an amino acidsequence described below. Preferably, these peptides have a bindingability to an HLA molecule. Also, these peptides preferably induce acytotoxic activity. Moreover, these peptides are preferably anHLA-A*2402-restricted eEF2 peptide, an HLA-A*0201-restricted eEF2peptide, or an HLA-A*0206-restricted eEF2 peptide. Furthermore, thesepeptides preferably have a length of 9 to 30 amino acids. In addition,the present invention provides a pharmaceutical composition for thetreatment or prevention of cancer which comprises these peptides, use ofthese peptides for the production of a pharmaceutical for the treatmentor prevention of cancer, a method for the treatment or prevention ofcancer, which comprises administering these peptides to a subject, andothers.

Thus, in one aspect, the present invention provides anHLA-A*2402-restricted eEF2 peptide. Also, the present invention providesan HLA-A*2402-restricted eEF2 peptide for the treatment or prevention ofcancer in an HLA-A*2402-positive subject, as well as a pharmaceuticalcomposition containing the same. An exemplary HLA-A*2402-restricted eEF2peptide used in the present invention is a peptide having an amino acidsequence composed of contiguous amino acids derived from an eEF2 proteinor a peptide containing an amino acid sequence composed of contiguousamino acids derived from an eEF2 protein, wherein the above amino acidsequence is selected from the group consisting of: Arg Phe Tyr Ala PheGly Arg Val Phe (SEQ ID NO:4); Ala Phe Gly Arg Val Phe Ser Gly Leu (SEQID NO:5); Arg Phe Asp Val His Asp Val Thr Leu (SEQ ID NO:6); Ala Tyr LeuPro Val Asn Glu Ser Phe (SEQ ID NO:7); and an amino acid sequence havingsubstitution or deletion or addition of several amino acids, forexample, 1 to 9, preferably 1 to 5, 1 to 4, 1 to 3, more preferably 1 to2 amino acids, and still more preferably one amino acid in one of theabove amino acid sequences; but is not limited to these peptides. Also,a peptide may be contained wherein the above amino acid sequence isselected from the group consisting of amino acid sequences havingsubstitution, deletion, addition and/or insertion of several aminoacids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1 to 3, morepreferably 1 to 2 amino acids, and still more preferably one amino acidin one of the above amino acid sequences. In case an amino acid in theabove peptides is substituted, preferred substitution sites are an aminoacid at position 2 and/or position 9. A preferred example of an aminoacid at position 2 in the above peptides is Phe or Tyr. Also, apreferred example of an amino acid at position 9 in the above peptidesis Ile, Leu or Phe. Specific examples of such a modified-type peptideare peptides as shown in Table 12 or Table 13. A preferred eEF2 peptidein the present invention is Ala Tyr Leu Pro Val Asn Glu Ser Phe (SEQ IDNO:7). In this regard, however, it is essential for all the abovepeptides to retain a binding ability to the HLA-A*2402 molecule. In thepresent specification, a peptide retaining a binding ability to theHLA-A*2402 is referred to as an HLA-A*2402-restricted eEF2 peptide. Inaddition, the above peptides of the present invention may be used for asubject other than the HLA-A*2402-positive subject. Accordingly, thepresent invention provides a peptide containing any one of the aboveamino acid sequences, and a pharmaceutical composition containing thepeptide.

In another aspect, the present invention provides anHLA-A*0201-restricted eEF2 peptide. Also, the present invention providesa pharmaceutical composition for the treatment or prevention of cancerin an HLA-A*0201-positive subject, comprising the HLA-A*0201-restrictedeEF2 peptide. The HLA-A*0201-restricted eEF2 peptide used in the presentinvention is a peptide having an amino acid sequence composed ofcontiguous amino acids derived from an eEF2 protein or a peptidecontaining an amino acid sequence composed of contiguous amino acidsderived from an eEF2 protein. Candidates of the HLA-A*0201-restrictedeEF2 peptide used in the present invention are exemplified in thefollowing Tables 1 to 7. Among them, an example of a preferred peptidein the present invention is a peptide wherein the above amino acidsequence is selected from the group consisting of:

Arg Leu Met Glu Pro Ile Tyr Leu Val (SEQ ID NO:8); Lys Leu Val Glu GlyLeu Lys Arg Leu (SEQ ID NO:9); Tyr Leu Asn Glu Ile Lys Asp Ser Val (SEQID NO:10); Ile Leu Thr Asp Ile Thr Lys Gly Val (SEQ ID NO:11); Leu MetMet Tyr Ile Ser Lys Met Val (SEQ ID NO:12); Lys Leu Pro Arg Thr Phe CysGln Leu (SEQ ID NO:13); Leu Ile Leu Asp Pro Ile Phe Lys Val (SEQ IDNO:14); and

an amino acid sequence having substitution or deletion or addition ofseveral amino acids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1to 3, more preferably 1 to 2 amino acids, and still more preferably oneamino acid in one of the above amino acid sequences; but is not limitedto these peptides. Also, a peptide may be contained wherein the aboveamino acid sequence is selected from the group consisting of amino acidsequences having substitution, deletion, addition and/or insertion ofseveral amino acids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1to 3, more preferably 1 to 2 amino acids, and still more preferably oneamino acid in one of the above amino acid sequences. Among them, aparticularly preferred HLA-A*0201-restricted eEF2 peptide is Arg Leu MetGlu Pro Ile Tyr Leu Val (SEQ ID NO:8) or Ile Leu Thr Asp Ile Thr Lys GlyVal (SEQ ID NO:11). Also, the HLA-A*0201-restricted eEF2 peptide of thepresent invention may have a substitution of an amino acid,particularly, at position 2 and/or at position 9 with another aminoacid. A preferred example of the amino acid at position 2 in the abovepeptides is Leu or Met. Also, a preferred example of the amino acid atposition 9 in the above peptides is Leu or Val. Examples of such amodified-type peptide are shown in Tables 15 to 21. Preferred examplesare peptides having, in the Leu Ile Leu Asp Pro Ile Phe Lys Val (SEQ IDNO:14), a substitution of the amino acid Ile at position 2 with Leu orMet, and/or a substitution of the amino acid Val at position 9 with Leu.Particularly preferred examples are Leu Leu Leu Asp Pro Ile Phe Lys Val(SEQ ID NO:15), Leu Met Leu Asp Pro Ile Phe Lys Val (SEQ ID NO:16), LeuLeu Leu Asp Pro Ile Phe Lys Leu (SEQ ID NO:17), or Leu Met Leu Asp ProIle Phe Lys Leu (SEQ ID NO:24). In this regard, however, it is essentialfor all the above peptides to retain a binding ability to the HLA-A*0201molecule. In the present specification, a peptide retaining a bindingability to the HLA-A*0201 is referred to as an HLA-A*0201-restrictedeEF2 peptide. Also, the HLA-A*0201-restricted eEF2 peptide of thepresent invention may have an action of increasing an interferon-γactivity. In addition, the above peptides of the present invention maybe used for a subject other than the HLA-A*0201-positive subject.Accordingly, the present invention provides a peptide containing any oneof the above amino acid sequences, and a pharmaceutical compositioncontaining the peptide.

In still another aspect, the present invention provides anHLA-A*0206-restricted eEF2 peptide. Also, the present invention providesa pharmaceutical composition for the treatment or prevention of cancerin an HLA-A*0206-positive subject which comprises theHLA-A*0206-restricted eEF2 peptide. The HLA-A*0206-restricted eEF2peptide used in the present invention is a peptide having an amino acidsequence composed of contiguous amino acids derived from an eEF2 proteinor a peptide containing an amino acid sequence composed of contiguousamino acids derived from an eEF2 protein. A preferred example of theHLA-A*0206-restricted eEF2 peptide used in the present invention is apeptide wherein the above amino acid sequence is selected from the groupconsisting of:

Arg Leu Met Glu Pro Ile Tyr Leu Val (SEQ ID NO:8); Lys Leu Val Glu GlyLeu Lys Arg Leu (SEQ ID NO:9); Tyr Leu Asn Glu Ile Lys Asp Ser Val (SEQID NO:10); Ile Leu Thr Asp Ile Thr Lys Gly Val (SEQ ID NO:11); Leu MetMet Tyr Ile Ser Lys Met Val (SEQ ID NO:12); Lys Leu Pro Arg Thr Phe CysGln Leu (SEQ ID NO:13); Leu Ile Leu Asp Pro Ile Phe Lys Val (SEQ IDNO:14); and

an amino acid sequence having substitution or deletion or addition ofseveral amino acids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1to 3, more preferably 1 to 2 amino acids, and still more preferably oneamino acid in one of the above amino acid sequences; but is not limitedto these peptides. Also, a peptide may be contained wherein the aboveamino acid sequence is selected from the group consisting of amino acidsequences having substitution, deletion, addition and/or insertion ofseveral amino acids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1to 3, more preferably 1 to 2 amino acids, and still more preferably oneamino acid in one of the above amino acid sequences. In this regard,however, it is essential for all the above peptides to retain a bindingability to the HLA-A*0206 molecule. In the present specification, apeptide retaining a binding ability to the HLA-A*0206 is referred to asan HLA-A*0206-restricted eEF2 peptide. Also, the HLA-A*0206-restrictedeEF2 peptide of the present invention may have an action of increasingan interferon-γ activity. In addition, the above peptides of the presentinvention may be used for a subject other than the HLA-A*0206-positivesubject. Accordingly, the present invention provides a peptidecontaining any one of the above amino acid sequences, and apharmaceutical composition containing the peptide.

TABLE 1 Starting residue Candidate number (Amino acid peptideresidue number in number Amino acid sequence SEQ ID NO: 1)  1 LILDPIFKV292 (SEQ ID NO: 14)  2 RLMEPIYLV 739 (SEQ ID NO: 8)  3 KLVEGLKRL 519(SEQ ID NO: 9)  4 YLNEIKDSV 671 (SEQ ID NO: 10)  5 ILTDITKGV 661(SEQ ID NO: 11)  6 LMMYISKMV 394 (SEQ ID NO: 12)  7 KLPRTFCQL 284(SEQ ID NO: 13)  8 GLHGWAFTL 217 (SEQ ID NO: 50)  9 GLVGVDQFL 471(SEQ ID NO: 51) 10 WLPAGDALL 343 (SEQ ID NO: 52) 11 VVVDCVSGV 127(SEQ ID NO: 53) 12 AIAERIKPV 146 (SEQ ID NO: 54) 13 IMIDPVLGT 203(SEQ ID NO: 55) 14 RLAKSDPMV 526 (SEQ ID NO: 56) 15 GLVSTGLKV 419(SEQ ID NO: 57)

TABLE 2 Starting residue Candidate number (Amino acid peptideresidue number in number Amino acid sequence SEQ ID NO: 1) 16 LVGVDQFLA472 (SEQ ID NO: 58) 17 KMDRALLEL 159 (SEQ ID NO: 59) 18 FVVKAYLPV 782(SEQ ID NO: 60) 19 TILMMGRYV 450 (SEQ ID NO: 61) 20 NLIDSPGHV 101(SEQ ID NO: 62) 21 ALDNFLDKL 850 (SEQ ID NO: 63) 22 CLYASVLTA 728(SEQ ID NO: 64) 23 LLQMITIHL 350 (SEQ ID NO: 65) 24 QVAGTPMFV 775(SEQ ID NO: 66) 25 VVAGFQWAT 679 (SEQ ID NO: 67) 26 LMMNKMDRA 155(SEQ ID NO: 68) 27 NMRVMKFSV 696 (SEQ ID NO: 69) 28 NMRVMKFSV 493(SEQ ID NO: 70) 29 AIMDKKANI  11 (SEQ ID NO: 71) 30 CVFDWQIL 812(SEQ ID NO: 72)

TABLE 3 Starting residue Candidate number (Amino acid peptideresidue number in number Amino acid sequence SEQ ID NO: 1) 31 GIPALDNFL847 (SEQ ID NO: 73) 32 VLNRKRGHV 762 (SEQ ID NO: 74) 33 MMGRYVEPI 453(SEQ ID NO: 75) 34 FLVKTGTIT 478 (SEQ ID NO: 76) 35 QVVGGIYGV 754(SEQ ID NO: 77) 36 RVTDGALVV 120 (SEQ ID NO: 78) 37 FQWATKEGA 683(SEQ ID NO: 79) 38 VAGTPMFVV 776 (SEQ ID NO: 80) 39 GLKEGIPAL 843(SEQ ID NO: 81) 40 SVLTAQPRL 732 (SEQ ID NO: 82) 41 PMFVVKAYL 780(SEQ ID NO: 83) 42 VMKFSVSPV 496 (SEQ ID NO: 84) 43 WAFTLKQFA 221(SEQ ID NO: 85) 44 FEHAHNMRV 488 (SEQ ID NO: 86) 45 KQFAEMYVA 226(SEQ ID NO: 87)

TABLE 4 Starting residue Candidate number (Amino acid peptideresidue number in number Amino acid sequence SEQ ID NO: 1) 46 RVFSGLVST415 (SEQ ID NO: 88) 47 RIVENVNVI 180 (SEQ ID NO: 89) 48 MMNKMDRAL 156(SEQ ID NO: 90) 49 EMYVAKFAA 230 (SEQ ID NO: 91) 50 FSVSPVVRV 499(SEQ ID NO: 92) 51 ELYQTFQRI 173 (SEQ ID NO: 93) 52 SVVAGFQWA 678(SEQ ID NO: 94) 53 IMNFKKEET 304 (SEQ ID NO: 95) 54 GALVVVDCV 124(SEQ ID NO: 96) 55 KVEDMMKKL 252 (SEQ ID NO: 97) 56 RNMSVIAHV  20(SEQ ID NO: 98) 57 KANIRNMSV  16 (SEQ ID NO: 99) 58 TVSEESNVL 582(SEQ ID NO: 100) 59 GVCVQTETV 134 (SEQ ID NO: 101) 60 DITKGVQYL 664(SEQ ID NO: 102)

TABLE 5 Starting residue Can- number (Amino didate acid residue peptidenumber in number Amino acid sequence SEQ ID NO: 1) 61AVMRRWLPA (SEQ ID NO: 103) 338 62 FSSEVTAAL (SEQ ID NO: 104) 111 63KLWGDRYFD (SEQ ID NO: 105) 259 64 LEPEELYQT (SEQ ID NO: 106) 169 65GVDQFLVKT (SEQ ID NO: 107) 474 66 FTLKQFAEM (SEQ ID NO: 108) 223 67AEMYVAKFA (SEQ ID NO: 109) 229 68 FTADLRSNT (SEQ ID NO: 110) 796 69MIDPVLGTV (SEQ ID NO: 111) 204 70 YLPVNESFG (SEQ ID NO: 112) 787 71NPADLPKLV (SEQ ID NO: 113) 513 72 GPAERAKKV (SEQ ID NO: 114) 245 73DLPKLVEGL (SEQ ID NO: 115) 516 74 MVNFTVDQI (SEQ ID NO: 116)   1 75GGQAFPQCV (SEQ ID NO: 117) 805

TABLE 6 Starting residue Can- number (Amino didate acid residue peptidenumber in number Amino acid sequence SEQ ID NO: 1) 76VLTAQPRLM (SEQ ID NO: 118) 733 77 SGLHGWAFT (SEQ ID NO: 119) 216 78ITIHLPSPV (SEQ ID NO: 120) 354 79 KSTLTDSLV (SEQ ID NO: 121)  32 80GELHLEICL (SEQ ID NO: 122) 550 81 CITIKSTAI (SEQ ID NO: 123)  67 82SEVTAALRV (SEQ ID NO: 124) 113 83 FTVDQIRAI (SEQ ID NO: 125)   4 84AQPRLMEPI (SEQ ID NO: 126) 736 85 YLAEKYEWD (SEQ ID NO: 127) 634 86KIWCFGPDG (SEQ ID NO: 128) 648 87 GTVGFGSGL (SEQ ID NO: 129) 210 88VEIQCPEQV (SEQ ID NO: 130) 747 89 KNPADLPKL (SEQ ID NO: 131) 512 90GVRFDVHDV (SEQ ID NO: 132) 699

TABLE 7 Starting residue Can- number (Amino didate acid residue peptidenumber in number Amino acid sequence SEQ ID NO: 1) 91TTFEHAHNM (SEQ ID NO: 133) 486 92 GNIVGLVGV (SEQ ID NO: 134) 467 93IIPTARRCL (SEQ ID NO: 135) 721 94 PLMMYISKM (SEQ ID NO: 136) 393 95GQLGPAERA (SEQ ID NO: 137) 242 96 LKQFAEMYV (SEQ ID NO: 138) 225 97MGNIMIDPV (SEQ ID NO: 139) 200 98 KVFDAIMNF (SEQ ID NO: 140) 299 99MEPIYLVEI (SEQ ID NO: 141) 741

The peptide used in the present invention is derived from an eEF2protein, and may consist of the above contiguous amino acid sequence ora modified sequence thereof, or contain such a sequence. In case thepeptide contains the above amino acid sequence, there is no particularlimitation on the length of the peptide, and the peptide may have anylength. Preferred examples of the peptide containing the abovecontiguous amino acid sequence are peptides having 9 to 30 amino acids,preferably peptides having 9 to 15 amino acids, and more preferablypeptides having 9 to 12 amino acids. Thus, the peptide used in thepresent invention may be, for example, the peptide itself consisting ofthe above amino acid sequence, or an eEF2 protein containing the aboveamino acid sequence or a portion thereof. In a peptide used in thepresent invention, a variety of substances can also be bound to an N-endand/or a C-end of a peptide containing the above amino acid sequence.For example, amino acids, peptides, and analogues thereof may be boundto the peptide. In case these substances are bound to a peptide used inthe present invention, they are treated, for example, by an enzyme andthe like in the body or through a process such as intracellularprocessing, and a peptide consisting of the above amino acid sequence isfinally produced. The peptide is presented on a cell surface as acomplex with an HLA-A*2402 molecule or HLA-A*0201 molecule, therebybeing able to produce an induction effect of cytotoxic T cells (CTL).These substances may be those which regulate solubility of a peptideused in the present invention, or improve stability of the peptide (e.g.protease-resistant effect), or, for example, specifically deliver apeptide used in the present invention to a given tissue or organ, orhave an enhancing action of an uptake efficiency of antigen-presentingcells and the like. Also, these substances may be a substance whichincreases an ability to induce the CTL, for example, a helper peptideand the like.

The peptide used in the present invention can be synthesized using amethod usually used in this art or a modified method thereof. Such asynthetic method is described, for example, in Peptide Synthesis,Interscience, New York, 1966; The Proteins, Vol. 2, Academic Press Inc.,New York, 1976; Peptide Synthesis, Maruzen Company Ltd., 1975; Basis andExperiment of Peptide Synthesis, Maruzen Company Ltd., 1985; Developmentof Medicine, Sequel, Vol. 14, Peptide Synthesis, Hirokawa Shoten Co.,1991 and others.

Also, the peptide used in the present invention can be prepared using agenetic engineering technique on the basis of the information on anucleotide sequence encoding the peptide used in the present invention.Such a genetic engineering technique is well known to those skilled inthe art.

The present invention relates to a pharmaceutical composition for thetreatment or prevention of cancer, comprising the above eEF2 peptide.Since the eEF2 gene is highly expressed, for example, in lungadenocarcinoma, small-cell lung cancer, esophageal cancer, stomachcancer, colon cancer, pancreatic duct cancer, malignant glioblastoma,malignant lymphoma, head-and-neck squamous cell cancer, and the like,the pharmaceutical composition of the present invention can be used forthe treatment or prevention of cancer expressing the eEF2 gene. When thepharmaceutical composition of the present invention is administered toan HLA-A*2402- or HLA-A*0201-positive subject, an eEF2-specific CTL isinduced by an HLA-A*2402-restricted eEF2 peptide or anHLA-A*0201-restricted eEF2 peptide contained in the pharmaceuticalcomposition, and cancer cells in the subject are impaired by such a CTL.

The pharmaceutical composition of the present invention may contain, forexample, carriers, excipients and the like, in addition to the aboveeEF2 peptide as an active ingredient. Since the HLA-A*2402-restrictedeEF2 peptide or HLA-A*0201-restricted eEF2 peptide contained in thepharmaceutical composition of the present invention induces aneEF2-specific CTL, the pharmaceutical composition of the presentinvention may contain a suitable adjuvant, or may be administeredtogether with a suitable adjuvant in order to enhance its inductionefficiency. Preferred adjuvants are, for example, a complete orincomplete Freund's adjuvant, aluminum hydroxide and the like, but arenot limited thereto. Also, the pharmaceutical composition of the presentinvention may contain a known peptide, for example, WT1 peptide and thelike, as an active ingredient, in addition to the above eEF2 peptide.

The administration method of the pharmaceutical composition of thepresent invention can be selected suitably depending on conditions suchas types of diseases, a state of subjects, and targeted sites. Themethod may be, for example, intradermal administration, subcutaneousadministration, intramuscular administration, intravenousadministration, transnasal administration, or oral administration, butis not limited thereto. Furthermore, the method may be a lymphocytetherapy or a DC (dendritic cell) therapy. The amount of a peptidecontained in the pharmaceutical composition of the present invention,dosage form of the pharmaceutical composition, number of doses and thelike may be selected suitably depending on conditions such as types ofdiseases, a state of subjects, and targeted sites. The amount of apeptide administered per one dose is usually from 0.0001 mg to 1000 mg,and preferably from 0.001 mg to 10,000 mg.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administering aneffective amount of the above pharmaceutical composition to anHLA-A*2402-positive subject or an HLA-A*0201-positive subject. Cancersto be treated or prevented may be any cancers, and include, for example,lung adenocarcinoma, non-small cell lung cancer, small-cell lung cancer,head-and-neck squamous cell cancer, esophageal squamous cell cancer,stomach cancer, colon cancer, pancreatic duct cancer, glioblastoma andmalignant lymphoma.

In still another aspect, the present invention relates to use of an eEF2peptide for the production of the above pharmaceutical composition.

In still another aspect, the present invention relates to apolynucleotide encoding the above eEF2 peptide (hereinafter, alsoreferred to as eEF2 polynucleotide). The polynucleotide of the presentinvention may be a DNA or an RNA. The base sequence of thepolynucleotide of the present invention can be determined on the basisof the amino acid sequence of the above eEF2 peptide. The abovepolynucleotide can be prepared, for example, by a DNA or RNA syntheticmethod, a PCR method and the like.

In another aspect, the present invention relates to an expression vectorcontaining the above polynucleotide (hereinafter, also referred to aseEF2 expression vector). The types of expression vectors, sequencescontained in addition to the above polynucleotide sequence and the likecan be selected suitably depending on types of hosts into which theexpression vector is introduced, the purpose of introducing theexpression vector and the like. The expression vector of the presentinvention is administered to a subject to produce an eEF2 peptide in aliving body and to induce an eEF2-specific CTL. The CTL impairshematopoietic organ tumor cells, solid cancer cells and the like in asubject, thereby allowing the hematopoietic organ tumor and solid cancerto be treated or prevented.

In still another aspect, the present invention relates to apharmaceutical composition for the treatment or prevention of cancer,comprising the above eEF2 polynucleotide or the above eEF2 expressionvector. Composition, administration method and the like of thepharmaceutical composition of the present invention in this aspect areas described above.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administering apharmaceutical composition containing an effective amount of the aboveeEF2 polynucleotide or eEF2 expression vector to a subject. Cancers tobe treated or prevented include, for example, lung adenocarcinoma,non-small cell lung cancer, small-cell lung cancer, head-and-necksquamous cell cancer, esophageal squamous cell cancer, stomach cancer,colon cancer, pancreatic duct cancer, glioblastoma, malignant lymphomaand the like.

In still another aspect, the present invention relates to use of an eEF2polynucleotide or an eEF2 expression vector for the production of apharmaceutical composition containing the above eEF2 polynucleotide oreEF2 expression vector.

In another aspect, the present invention relates to cells containing theabove eEF2 expression vector. The cells of the present invention can beprepared, for example, by transforming host cells such as E. coli,yeast, insect, and animal cells using the above expression vector. Amethod for introducing the expression vector into the host cells can beselected suitably from various methods. It is also possible to preparethe peptide of the present invention by culturing transformed cells, andrecovering and purifying an eEF2 peptide produced.

In a further aspect, the present invention relates to an eEF2-specificCTL which is induced by the above eEF2 peptide. The CTL of the presentinvention recognizes a complex of an eEF2 peptide with an HLA-A*2402molecule or an HLA-A*0201 molecule. Accordingly, HLA-A*2402-positive orHLA-A*0201-positive and highly eEF2-expressing tumor cells can beimpaired specifically using the CTL of the present invention.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administering aneEF2-specific CTL to an HLA-A*2402 positive or HLA-A*0201-positivesubject. The administration method of the eEF2-specific CTL can beselected suitably depending on conditions such as types of diseases, astate of subjects, and targeted sites. The method may be, for example,intravenous administration, intradermal administration, subcutaneousadministration, intramuscular administration, transnasal administration,or oral administration, but is not limited thereto.

In another aspect, the present invention relates to a method forinducing an eEF2-specific CTL, which comprises culturing peripheralblood mononuclear cells in the presence of the aboveHLA-A*2402-restricted eEF2 peptide or HLA-A*0201-restricted eEF2peptide, and the eEF2-specific CTL is induced from the peripheral bloodmononuclear cells. Subjects from which the peripheral blood mononuclearcells are derived may be any subjects so far as they have an HLA-A*2402molecule or an HLA-A*0201 molecule. By culturing the peripheral bloodmononuclear cells in the presence of the HLA-A*2402-restricted eEF2peptide or HLA-A*0201-restricted eEF2 peptide, the eEF2-specific CTL isinduced from CTL precursor cells in the peripheral blood mononuclearcells. By administering the eEF2-specific CTL obtained by the presentinvention to an HLA-A*2402-positive subject or an HLA-A*0201-positivesubject, it is possible to treat or prevent a hematopoietic organ tumorand a solid cancer in the subject. In this connection, the peripheralblood mononuclear cells in the present specification include immatureantigen-presenting cells (for example, precursors of dendritic cells,B-lymphocytes, macrophages, etc.) which are precursors ofantigen-presenting cells. Since the immature antigen-presenting cellsare contained, for example, in peripheral blood mononuclear cells andthe like, such cells may be cultured in the presence of the above eEF2peptide.

In still another aspect, the present invention relates to a kit forinducing an eEF2-specific CTL, comprising an HLA-A*2402-restricted eEF2peptide or an HLA-A*0201-restricted eEF2 peptide as an essentialconstituent. Preferably, the kit is used in a method for inducing theabove eEF2-specific CTL. The kit of the present invention may contain,for example, a sampling means of peripheral blood mononuclear cells, anadjuvant, a reaction vessel and the like, in addition to the aboveHLA-A*2402-restricted eEF2 peptide or HLA-A*0201-restricted eEF2peptide. In general, the kit is accompanied with an instruction manual.The kit of the present invention can be used to induce efficiently theeEF2-specific CTL.

In a further aspect, the present invention relates to antigen-presentingcells (for example, dendritic cells, B-lymphocytes, macrophages, etc.)which present the above eEF2 peptide through an HLA-A*2402 molecule oran HLA-A*0201 molecule. The antigen-presenting cells of the presentinvention are induced by the above HLA-A*2402-restricted eEF2 peptide orHLA-A*0201-restricted eEF2 peptide. The above eEF2-specific CTL isefficiently induced using the antigen-presenting cells of the presentinvention.

In another aspect, the present invention relates to a method for thetreatment or prevention of cancer, which comprises administeringantigen-presenting cells, which present the above eEF2 peptide throughan HLA-A*2402 molecule or an HLA-A*0201 molecule, to anHLA-A*2402-positive subject or an HLA-A*0201-positive subject. Theadministration method of the antigen-presenting cells can be selectedsuitably depending on conditions such as types of diseases, a state ofsubjects, and targeted sites. The method may be, for example,intravenous administration, intradermal administration, subcutaneousadministration, intramuscular administration, transnasal administration,or oral administration, but is not limited thereto.

In a further aspect, the present invention relates to a method forpreventing or treating cancer, which comprises inducingantigen-presenting cells which present an eEF2 peptide through anHLA-A*2402 molecule or an HLA-A*0201 molecule, the method comprising thesteps of:

(a) reacting a sample with a nucleic acid having a nucleotide sequenceencoding an amino acid sequence (SEQ ID NO:1) of an eEF2 protein or apartial sequence thereof, or the above eEF2 peptide,

(b) obtaining antigen-presenting cells which present the eEF2 peptidecontained in the sample through the HLA-A*2402 molecule or HLA-A*0201molecule, and

(c) administering the antigen-presenting cells to an HLA-A*2402-positivesubject or an HLA-A*0201-positive subject. The sample in the abovemethod may be any samples so far as they have a possibility of inclusionof lymphocytes or dendritic cells, and includes, for example, samplesfrom a subject such as blood, cell culture media and the like. Thereaction in the above method may be carried out using a conventionaltechnique, preferably using an electroporation technique. The obtainmentof the antigen-presenting cells can be carried out using a method knownto those skilled in the art. The culture conditions of cells in a samplein each step can be determined suitably by those skilled in the art. Theadministration method of the antigen-presenting cells may be asdescribed above.

Furthermore, the present invention relates to a kit for preventing ortreating cancer, comprising, as an essential constituent, a nucleic acidhaving a nucleotide sequence encoding an amino acid sequence (SEQ IDNO:1) of an eEF2 protein or a partial sequence thereof, or an eEF2peptide. The kit comprises antigen-presenting cells which present theabove eEF2 peptide through an HLA-A*2402 molecule or an HLA-A*0201molecule. Also, the kit of the present invention may contain, forexample, a sampling means, a reaction vessel and the like, in additionto the above essential constituent. In general, the kit is accompaniedwith an instruction manual. The antigen-presenting cells which presentan eEF2 peptide through an HLA-A*2402 molecule or an HLA-A*0201 moleculecan be obtained efficiently using the kit of the present invention, andcancer can be treated or prevented by administering theantigen-presenting cells.

In another aspect, the present invention relates to an antibody againstan HLA-A*2402-restricted eEF2 peptide or an HLA-A*0201-restricted eEF2peptide, or an antibody against a polynucleotide encoding the peptide.The antibody of the present invention may be either a polyclonalantibody or a monoclonal antibody.

In a further aspect, the present invention relates to a method fordiagnosing cancer, characterized by the use of the above eEF2-specificCTL, antigen-presenting cells which present the above eEF2 peptidethrough an HLA-A*2402 molecule or an HLA-A*0201 molecule, or an antibodyagainst an HLA-A*2402-restricted eEF2 peptide or anHLA-A*0201-restricted eEF2 peptide or an antibody against apolynucleotide encoding the peptide. The eEF2-specific CTL is preferablyused in the diagnosis method of the present invention. For example,cancer can be diagnosed by incubating the above CTL, antigen-presentingcells or antibody with a sample from an HLA-A*2402-positive subject oran HLA-A*0201-positive subject, or administering the above CTL,antigen-presenting cells or antibody to an HLA-A*2402-positive subjector an HLA-A*0201-positive subject, and then determining, for example,the position, site, amount or the like of the CTL, antigen-presentingcells or antibody. The above CTL, antigen-presenting cells or antibodymay be labeled. By such labeling, the diagnosis method of the presentinvention can be carried out efficiently.

In another aspect, the present invention relates to a kit for diagnosisof cancer, comprising, as an essential constituent, the aboveeEF2-specific CTL, antigen-presenting cells which present an eEF2peptide through an HLA-A*2402 molecule or an HLA-A*0201 molecule, or anantibody against an HLA-A*2402-restricted eEF2 peptide or anHLA-A*0201-restricted eEF2 peptide or an antibody against apolynucleotide encoding the peptide.

In a further aspect, the present invention relates to a method fordetermining the presence or amount of an eEF2-specific CTL in anHLA-A*2402-positive subject or an HLA-A*0201-positive subject, whichcomprises the steps of:

(a) reacting a complex of an eEF2 peptide and an HLA-A*2402 molecule oran HLA-A*0201 molecule with a sample from the subject, and then

(b) determining the presence or amount of the CTL recognizing thecomplex contained in the sample.

The sample from the subject may be any samples so far as they have apossibility of inclusion of lymphocytes, and includes, for example, bodyfluid such as blood and lymph fluid, tissues and the like. The complexof an eEF2 peptide and an HLA-A*2402 molecule or an HLA-A*0201 moleculemay be, for example, in the form of a tetramer, pentamer and the like,for example, using a method known to those skilled in the art such as abiotin-streptavidin method. The presence or amount of the CTLrecognizing such a complex can be determined by a method known to thoseskilled in the art. In this aspect of the present invention, the abovecomplex may be labeled. A known label such as a fluorescence label and aradioactive label can be used. By such labeling, the presence or amountof the CTL can be determined easily and rapidly. This aspect of themethod of the present invention allows diagnosis of cancer, prognosticdiagnosis and the like.

Thus, the present invention also provides a composition comprising acomplex of an eEF2 peptide and an HLA-A*2402 molecule or an HLA-A*0201molecule for determining the presence or amount of an eEF2-specific CTLin an HLA-A*2402-positive subject or an HLA-A*0201-positive subject.

Also, the present invention provides a kit for determining the presenceor amount of an eEF2-specific CTL in an HLA-A*2402-positive subject oran HLA-A*0201-positive subject, comprising a complex of an eEF2 peptideand an HLA-A*2402 molecule or an HLA-A*0201 molecule.

In a further aspect, the present invention relates to a method forobtaining an eEF2-specific CTL using a complex of an eEF2 peptide and anHLA-A*2402 molecule or an HLA-A*0201 molecule, which comprises the stepsof:

(a) reacting a sample with the complex, and

(b) obtaining the CTL which recognizes the complex contained in thesample.

The complex of an eEF2 peptide and an HLA-A*2402 molecule or anHLA-A*0201 molecule is as described above. The sample may be any samplesso far as they have a possibility of inclusion of lymphocytes, andincludes, for example, samples from a subject such as blood, cellculture media and the like. The obtainment of the CTL recognizing thecomplex can be carried out using a method known to those skilled in theart, for example, using FACS, MACS and the like. The eEF2-specific CTLobtained can be cultured to use for the treatment or prevention of avariety of cancers.

Thus, the present invention also relates to an eEF2-specific CTL, whichcan be obtained by a method for obtaining the eEF2-specific CTL using acomplex of an eEF2 peptide and an HLA-A*2402 molecule or an HLA-A*0201molecule.

Furthermore, the present invention relates to a kit for obtaining aneEF2-specific CTL, comprising a complex of an eEF2 peptide and anHLA-A*2402 molecule or an HLA-A*0201 molecule.

In one aspect, the present invention provides a kit for tumorigenesischaracterized in that an eEF2 polypeptide is expressed. Thus, the kit ofthe present invention comprises, as an essential constituent, the stepof expressing the eEF2 polypeptide in cells or non-human animals.Accordingly, a polynucleotide encoding an amino acid sequence of thepolypeptide or a vector into which the polynucleotide is integrated isan essential constituent. In the present specification, the non-humananimals refer to animals other than human. The kit of the presentinvention is based on a finding that forced expression of an eEF2protein accelerates a G2/M phase in a cell cycle. The kit of the presentinvention may also contain, for example, a means for introducing theabove polynucleotide or vector into cells or non-human animal tissues, areagent for introduction, a reaction vessel and the like, in addition tothe above polynucleotide or vector. In general, the kit is accompaniedwith an instruction manual. The kit of the present invention can be usedfor forming a tumor in vivo or in vitro, and then, for testing effectsof candidate molecules against tumorigenesis or cell proliferation, forexample.

In this regard, the method of the present invention may be carried outin vivo or in vitro.

EXAMPLES

The present invention is illustrated particularly and specifically byreferring to the following examples, which should not be construed aslimiting the present invention.

Example 1 Detection of EEF2 IgG Antibody in Sera of Cancer Patients

Cells of lung cancer cell lines PC14 and LU-99B, and leukemia cell lineK562 were lysed in an SDS-sample buffer. Proteins contained in thebuffer were separated by SDS-PAGE, and then transferred to a PVDFmembrane. Solutions prepared by diluting sera obtained from 10 patientshaving lung cancer and 10 healthy subjects by 1500:1 were used as aprimary antibody, and IgG antibodies bound to the membrane werevisualized using an anti-human IgG antibody. As a result, a protein ofabout 100 kDa was found which was specifically recognized by the serafrom the patients having lung cancer (FIG. 1). FIG. 1 is a typicalexample of a western blot. Subsequently, the protein was separated andidentified as eEF2 by a mass spectrometric technique.

Excessive Expression of EEF2 in Various Cancers

Thin-sliced sections were prepared from paraffin-embedded blocks. Afterde-paraffin treatment, the sections were subjected to antigen-activatingtreatment in a citrate buffer (pH 6.0), reacted with an anti-eEF2antibody (H-118, Santa Cruz Biotechnology, Santa Cruz, Calif., 1:100dilution) at 4° C. overnight, and then reacted with Envision kit/HRP(Dako Cytomation) at room temperature for 30 minutes. After reactingwith 0.7% of an H₂O₂ solution, the sections were color-developed usingDAB as a substrate, and nuclear-staining was then carried out usinghematoxylin. As a result, antibody-positive cells were observed in eachaffected tissue of patients having lung adenocarcinoma, small-cell lungcancer, head-and-neck squamous cell cancer, esophageal cancer, stomachcancer and colon cancer (FIGS. 2 to 4).

Detection of EEF2 Antibody in Various Types of Cancers

Peripheral blood was obtained from 72 patients having non-small celllung cancer, 42 patients having colon cancer, 20 patients havinghead-and-neck squamous cell cancer, 18 patients having glioblastoma, and17 healthy subjects in agreement. The blood was coagulated and sera werethen obtained by centrifugation. A vector pGEX-5X-3 (GE) for expressionof a recombinant protein was prepared by inserting a gene sequenceencoding an amino acid sequence at positions 411-858 of eEF2. Therecombinant GST-eEF2₄₁₁₋₈₅₈ protein purified was adjusted to 150 ng/laneand SDS-PAGE was carried out. The protein on the SDS-PAGE waselectrically transferred to a PVDF membrane. The protein was reactedwith sera diluted by 1500:1 at room temperature overnight, and IgGantibodies bound to the membrane were visualized using an anti-human IgGantibody. Density of the bands was measured and used as an anti-eEF2antibody titer. Since the median value of the antibody titer in 17healthy subjects was 500 densitometric units and the standard deviationwas 500, the cutoff level was set to 2,000 densitometric units which wasmedian+3 SD. As a result, it was found that, at a specificity of 94.7%,the eEF2 IgG antibody was positive in 66.7% of non-small cell lungcancer, 71.8% of colon cancer, 60.0% of head-and-neck squamous cellcancer, and 88.9% of glioblastoma (FIG. 5). Expression of the eEF2protein in various cancers was analyzed by immunostaining. When thepercentage of cancer cells showing intense stain as compared withcorresponding normal cells was 25% or more of total cancer cells, theexpression was judged to be positive (Table 8).

TABLE 8 Excessive expression of eEF2 in various cancers Positive rate ofexcessive Cancer expression of eEF2 Lung adenocarcinoma  100% (15/15)Small-cell lung cancer 95.0% (19/20) Esophageal cancer 58.3% (7/12) Stomack cancer 92.9% (13/14) Colon cancer 91.7% (22/24) Pancreatic ductcancer 55.6% (5/9)  Malignant glioblastoma 50.6% (6/12)  Malignantlymphoma 94.0% (47/50) Head-and-neck squamous cell cancer 45.5% (5/11) 

Early Stage Detection of Cancer by EEF2 Antibody

The positive rate of eEF2 antibody titer was compared with the positiverate of CEA in 70 patients (44 in stage I, 13 in stage II, and 13 instage III) having non-small cell lung cancer and having a clear serumCEA value. In this connection, the classification of stage I, stage II,and stage III was carried out according to the TNM classificationdefined by the International Union Against Cancer. The eEF2 antibodytiter was determined by dot blotting. As a result, it was found that thepositive rate of the eEF2 IgG antibody in each disease stage ofnon-small cell lung cancer was high even from stage I (Table 9).

TABLE 9 Stage I II III Anti-eEF2 IgG antibody Positive rate 81.8% 61.5%61.5% CEA Positive rate 13.6% 23.1% 46.2%

Relationship Between EEF2 Antibody Titer and Disease-Free Survival Rate

Relationship between the eEF2 antibody titer in non-small cell lungcancer and disease-free survival rate was analyzed. Among 44 patients,the group (11 patients) having an eEF2 antibody titer of 4,000 or moredensitometric units has a significantly high disease-free survival rateas compared with the group (26 patients) having an eEF2 antibody titerof 2,000 to 4,000 densitometric units and the group (7 patients) havingan eEF2 antibody titer of less than 2,000 densitometric units (log ranktest, FIG. 6).

Example 2 Inhibition of Cell Proliferation in Various Cell Lines

A vector expressing a siRNA which targets at sequence:5′-caugggcaacaucaugaucgauccuguccu-3′ of an eEF2 mRNA (hereinafter,referred to as shEF2) was prepared using a tRNA-shRNA expression vector,piGENE tRNA Pur (Clontech, Palo Alto, Calif.). Subsequently, 10 μg ofshEF2 or a vacant shRNA vector (shMock) was introduced byelectroporation into stomach cancer cell lines AZ-521 and MKN28, coloncancer cell line SW620, lung cancer cell lines LU99B and PC-14,pancreatic cancer cell lines MiaPaCa2 and PCI6, glioblastoma cell linesA172 and U87MG, as well as malignant lymphoma cell lines IB4 and YT(each 5×10⁵ cells) expressing the eEF2 using Gene Pulser Xcell(trademark) system (Bio Rad, Hercules, Calif.) under a condition of 165V and 1000 hF. After 24, 48, 72 and 96 hours of the introduction, cellswere treated with trypsin and the number of surviving cells was counted.The experiments were carried out separately 3 times in duplicate. In allcases, the shEF2 significantly inhibited cell proliferation (FIGS. 7 and8).

Example 3 Selection of EEF2 Peptide

Firstly, 4 peptides were selected by predicting sequences which can bindto an HLA-A*2402 molecule in an amino acid sequence of an eEF2 proteinusing ProPred-I website. The results are shown in Table 10 below.

TABLE 10 (Table 10: Candidate eEF2 peptides having a high bindingaffinity to HLA-A*2402 molecule, selected using various programs(NetMHC3.0, Rankpep and SYFPEITHI)) NetMHC3.0 Rankpep SYFPEITHI StartingStarting Starting residue Affinity Binding Log residue residue number(nM) level score number Score number Score 786 20 SB 0.721 633 18.484786 25 633 188 WB 0.516 342 17.792 78 20 220 380 WB 0.451 477 15.220 26519 342 416 WB 0.442 786 14.930 477 19 477 671 0.398 174 13.101 412 18409 964 0.365 817 11.358 701 17 174 1130 0.350 684 11.215 409 16 6841250 0.341 220 10.709 308 15 177 1611 0.317 409 10.641 311 15 265 19300.301 701 9.634 470 15 78 1958 0.299 714 9.556 512 15 412 2551 0.275 789.315 516 15 231 2665 0.271 412 8.827 594 15 729 2691 0.270 443 8.652 7314 744 2829 0.265 364 8.246 252 14 73 3045 0.259 456 8.229 284 14 704080 0.232 213 8.072 328 14 644 4396 0.225 605 7.603 343 14 701 53220.207 265 7.401 434 14 759 6139 0.194 363 7.190 442 14 638 6593 4917.188 456 14 602 6673 177 6.847 491 14 396 7134 442 6.419 509 14 2847142 73 6.112 537 14 774 7954 850 6.015 657 14 736 8076 293 5.071 62 13442 8141 166 4.961 70 13 290 8704 763 4.815 92 13 394 8917 670 4.451 9513 300 8973 290 4.265 111 13 456 9299 285 4.229 180 13 227 9749 90 4.062191 13 180 9862 335 4.016 201 13 578 9900 396 3.935 228 13 264 10704 4533.878 258 13 491 11038 289 3.877 277 13 529 11225 1 3.85 293 13 29312329 744 3.694 296 13 811 12728 649 3.487 299 13 299 12938 38 3.281 30713

The starting residue number is the number shown in SEQ ID NO:1. Allcandidate eEF2 peptides are composed of 9 residues of amino acids. Forexample, a peptide having the starting residue number of 786 is apeptide composed of 9 amino acid residues from 786th residue A to 794thresidue F in SEQ ID NO:1.

Next, a binding ability to an HLA-A2402 molecule was actually analyzedby an MHC stabilization assay. Briefly, T2-2402 cells (1×10⁶ cells),receiving forced expression of a human HLA-A*2402 molecule, not havingan antigen-presenting ability to an HLA molecule, were incubated in anRPMI1640 medium containing 10 μM of a synthesized peptide and notcontaining a serum at 27° C. for 16 hours, and then allowed to stand at37° C. for 3 hours. Since expression of an HLA-A24 molecule on a cellsurface is stabilized by binding of a peptide, the expression of theHLA-A24 molecule on a cell surface after treatment with each peptide wasanalyzed by flow cytometry, and binding ability of each peptide to theHLA-A2402 molecule was evaluated. As a result, it was found thateEF2₄₀₉₋₄₁₇ (SEQ ID NO:4), eEF2₄₁₂₋₄₂₀ (SEQ ID NO:5), eEF2₇₀₁₋₇₀₉ (SEQID NO:6) and eEF2₇₈₆₋₇₉₄ (SEQ ID NO:7) peptides show a binding abilityto the HLA-A2402 molecule (Table 11).

TABLE 11 Identification of HLA-A2402-restricted eEF2 peptide Bindingability to eEF2 Peptide HLA-A2402 molecule eEF2₄₀₉₋₄₁₇ + eEF2₄₁₂₋₄₂₀ +eEF2₇₀₁₋₇₀₉ + eEF2₇₈₆₋₇₉₄ +

Determination of Interferon Activity

T cells were incubated together with HLA-A*2402 molecule-expressing T2cells pulsed with eEF2 peptides (eEF2₄₀₉₋₄₁₇, eEF2₄₁₂₋₄₂₀ andeEF2₇₀₁₋₇₀₉ peptides) in the presence of brefeldin A (Sigma) at 37° C.for 5 hours. After washing with PBS, CD3 and CD8 molecules which arecell surface antigens were stained by PerCP-conjugated anti-CD3 (BDBiosciences) and PE-conjugated anti-CD8 (Caltag, Burlingame, Calif.)antibodies on ice for 15 minutes. Subsequently, cells were fixed usingCytofix (BD Biosciences) on ice for 20 minutes, and intracellular IFN-γwas reacted with an FITC-conjugated anti-IFN-γ antibody (BD Biosciences)on ice for 30 minutes. Frequency of IFN-γ-positive cells present inCD8-positive T cells was analyzed using a flow cytometer. As a result,it was found that eEF2₄₀₉₋₄₁₇, eEF2₄₁₂₋₄₂₀ and eEF2₇₀₁₋₇₀₉ peptidesincrease the interferon-γ activity, and is therefore represent anHLA-A*2402-restricted peptide (FIG. 30).

Binding Affinity of Modified-type EEF2 Peptides to HLA-A*2402 Molecule

Furthermore, a binding affinity of modified-type eEF2 peptides, in whichan amino acid at position 2 (hereinafter, also referred to as P2) and/orat position 9 (hereinafter, also referred to as P9) in the amino acidsequences of eEF2₇₈₆₋₇₉₄ (SEQ ID NO:7) and eEF2₄₀₉₋₄₁₇ (SEQ ID NO:4)peptides among the above peptides was altered to another amino acid, waspredicted as described above.

TABLE 12 (Table 12: Prediction of binding affinity ofmodified-type eEF2₇₈₆₋₇₉₄ peptides (SEQ ID NOs:25 and 26) to HLA-A*2402 molecule) Can- Bind- didate Amino acid Bind-ing Log Peptide sequence ing level score Score 786 AYLPVNESF  20 SB0.721 14.930 786 I AYLPVNESI (SEQ  43 SB 0.652 15.164 ID NO: 25) 786 LAYLPVNESL (SEQ 143 WB 0.541 14.547 ID NO: 25)

TABLE 13 (Table 13: Prediction of binding affinity ofmodified-type eEF2₄₀₉₋₄₁₇ peptides (SEQ ID NOs:27 to 31) to HLA-A*2402 molecule) Can- Bind- didate Amino acid Bind- ingLog Peptide sequence ing level score Score 409 RFYAFGRVF 0.365 10.641 Y409 RFYAFGRVF (SEQ SB 0.641 15.653 ID NO: 27) 409 I RFYAFGRVI (SEQ 0.25210.875 NO: 28) Y 409 I RFYAFGRVI (SEQ WB 0.556 15.887 ID NO: 29) 409 LRFYAFGRVL (SEQ 0.164 10.258 NO: 30) Y 409 L RFYAFGRVL (SEQ WB 0.43415.270 ID NO: 31)

Since the eEF2₇₈₆₋₇₉₄ (SEQ ID NO:7) peptide has Y at P2 and F at P9 andthe Y and F are anchor residues, improvement of the binding affinity wasnot recognized even if the original residue is altered to anotherresidue (Table 12). On the other hand, remarkable improvement of thebinding affinity was recognized in the eEF2₄₀₉₋₄₁₇ (SEQ ID NO:4) peptidewhen the residue at P2 is altered to anchor residue Y (Table 13).

Induction of EEF2-Specific Killer T Cells

From peripheral blood mononuclear cells obtained from a donor having anHLA-A*2402 molecule, CD4⁺ CD25⁺ Treg cells were removed using CD25MicroBeads (Miltenyi Biotech, Auburn, Calif.). Subsequently, monocytesof the donor were isolated using BD IMag CD14 isolation kit (BDBioscience), and cultured in X-VIVO15 (Bio Whittaker, Walkersville, Md.)containing IL-4 and GM-CSF and supplemented with 1% human AB serum. Nextday, IL-1β, IL-6, TNF-α and PGE-2 were added for maturation of dendriticcells, and culture was continued for further 3 days. The dendritic cellswere irradiated (30 G), and then cultured in a medium containing 10μg/mL of a peptide for 2 hours to pulse the dendritic cells with apeptide. The mononuclear cells (2×10⁶ cells) having Treg cells removedwere then co-cultured with the dendritic cells pulsed with a peptide ina ratio of 10:1 to carry out stimulation by a peptide, and IL-2 wasadded to the medium on the next day. Subsequently, restimulation wascarried out every 10 days by the donor mononuclear cells irradiated andpulsed with a peptide. After carrying out several times of stimulation,the cells were cultured in a medium containing IL-7 and IL-15, and Tcell clones are established.

Determination of Cytotoxic Activity

From T cell clones established as described above, CD8-positive T cellswere purified using CD8 Microbeads to prepare effector cells.Subsequently, target cells were incubated with ⁵¹Cr-labeled sodiumchromate (Amersham Biosciences Corp., NJ) for 1 hour to label the cells.They were then mixed with the effector cells so that the ratio of cellcount was 1:1, 3:1 and 9:1 of CTL/target cell (E/T) ratio, and themixture was allowed to stand for 4 hours. The percentage of cells lysedwas calculated according to the following equation:

Specific lysis %=[(cpm experimental release−cpm spontaneousrelease)/(cpm maximal release−cpm spontaneous release)]×100.

T2-2402 cells pulsed with an eEF2₇₈₆₋₇₉₄ pep tide were used as thetarget cells, and T2-2402 cells not pulsed with the eEF2₇₈₆₋₇₉₄ peptide(SEQ ID NO:7) as a negative control. As a result, it was shown that thespecific lysis % of T2-2402 cells pulsed with the eEF2₇₈₆₋₇₉₄ peptideremarkably increases with the increase of the E/T ratio (FIG. 9). Also,colon cancer SW480 cells which show endogenous eEF2 expression and showHLA-A*2402 expression on a cell surface were used as the target cells,and stomach cancer AZ-521 cells and pancreatic cancer MiaPaCa2 cellswhich show endogenous eEF2 expression but do not show HLA-A*2402expression on a cell surface as a negative control. As a result, it wasshown that cytotoxic T cells activated by the eEF2₇₈₆₋₇₉₄ peptidespecifically impair the cells which express the eEF2 and have theHLA-A*2402 molecule (FIG. 10).

Example 4 Selection of EEF2 Peptide

Peptides were selected by predicting sequences which can bind to anHLA-A*0201 molecule in an amino acid sequence of an eEF2 protein usingProPred-I website (Tables 1 to 7). Next, a binding ability to anHLA-A0201 molecule was actually analyzed by an MHC stabilization assay.Briefly, T2-0201 cells (1×10⁶ cells), receiving forced expression of ahuman HLA-A*0201 molecule, not having an antigen-presenting ability toan HLA molecule, were incubated in an RPMI1640 medium containing 10 aMof a synthesized peptide and not containing a serum at 27° C. for 16hours, and then allowed to stand at 37° C. for 3 hours. Since expressionof an HLA-A*0201 molecule on a cell surface is stabilized by binding ofa peptide, the expression of the HLA-A0201 molecule on a cell surfaceafter treatment with each peptide was analyzed by flow cytometry, andbinding ability of each peptide to the HLA-A0201 molecule was evaluated.As a result, it was found that eEF2₂₈₄₋₂₉₂ (SEQ ID NO:13), eEF2₃₉₄₋₄₀₂(SEQ ID NO:12), eEF2₅₁₉₋₅₂₇ (SEQ ID NO:9), eEF2₆₆₁₋₆₆₉ (SEQ ID NG:11).eEF2₆₇₁₋₆₇₉ (SEQ ID NG:10) and eEF2₇₃₉₋₇₄₇ (SEQ ID NO:8) peptides show abinding ability to the HLA-A*0201 molecule (Table 14).

TABLE 14 Binding ability of candidate peptide toHLA-A0201 class I molecule Amino acid % MFI Candidate peptide sequenceMFI increase NS 5.8 Non-peptide 275.7 eEF2₂₉₂₋₃₀₀ (SEQ ID NO: 14)LILDPIFKV 781.03 183.3 eEF2₇₃₉₋₇₄₇ (SEQ ID NO: 8) RLMEPIYLV 664.83 141.1eEF2₅₁₉₋₅₂₇ (SEQ ID NO: 9) KLVEGLKRL 437.97 58.9eEF2₆₇₁₋₆₇₉ (SEQ ID NO: 10) YLNEIKDSV 522.71 89.6eEF2₆₆₁₋₆₆₉ (SEQ ID NO: 11) ILTDITKGV 828.16 200.4eEF2₃₉₄₋₄₀₂ (SEQ ID NO: 12) LMMYISKMV 448.41 62.6eEF2₂₈₄₋₂₉₂ (SEQ ID NO: 13) KLPRTFCQL 448.82 62.8

Determination of Interferon Activity

T cells were incubated together with HLA-A*0201 molecule-expressing T2cells pulsed with HLA-A*0201-restricted eEF2 peptides in the presence ofbrefeldin A (Sigma) at 37° C. for 5 hours. After washing with PBS, CD3and CD8 molecules which are cell surface antigens were stained byPerCP-conjugated anti-CD3 (BD Biosciences) and PE-conjugated anti-CD8(Caltag, Burlingame, Calif.) antibodies on ice for 15 minutes.Subsequently, cells were fixed using Cytofix (BD Biosciences) on ice for20 minutes, and intracellular IFN-γ was reacted with an FITC-conjugatedanti-IFN-γ antibody (BD Biosciences) on ice for 30 minutes. Frequency ofIFN-γ-positive cells present in CD8-positive T cells was analyzed usinga flow cytometer. An eEF2₇₃₉₋₇₄₇ peptide was used in FIG. 11, and aneEF2₆₆₁₋₆₆₉ peptide in FIG. 12. As a result, it was found thateEF2₆₆₁₋₆₆₉ (SEQ ID NO:11) and eEF2₇₃₉₋₇₄₇ (SEQ ID NO:8) peptidesincrease the interferon-γ activity (FIGS. 11 and 12).

Determination of Cytotoxic Activity

Next, evaluation was carried out as to whether the six candidatepeptides [eEF2₇₃₉₋₇₄₇ (SEQ ID NO:8), eEF2₈₁₉₋₅₂₇ (SEQ ID NO:9),eEF2₆₇₁₋₆₇₉ (SEQ ID NO:10), eEF2₆₆₁₋₆₆₉ (SEQ ID NO:11), eEF2₃₉₄₋₄₀₂ (SEQID NO:12) and eEF2₂₈₄₋₂₉₂ (SEQ ID NO:13), excepting eEF2₂₉₂₋₃₀₀ (SEQ IDNO:14)] selected as described above actually have a cytotoxic activity.The experiments were carried out in much the same way as in the aboveExample 3. Thus, blood was taken from healthy donors having anHLA-A*0201 molecule, peripheral blood mononuclear cells were separated,and the first stimulation was carried out using the six candidatepeptides (stimulator: self-PBMC). Subsequently, the peptide stimulationon the second day and later was carried out at intervals of 8 to 13 days(stimulator: allo B-LCL 3 mg/ml). Furthermore, IL-2 was added every 2days after the second stimulation at a final concentration of 20 IU/ml.Cytotoxicity was measured on the 6th day after the final peptidestimulation. As a result, increase of cytotoxic activity was observed inthe above 6 peptides (FIGS. 16 to 21). From the above fact, it was foundthat the above 6 peptides [eEF2₇₃₉₋₇₄₇ (SEQ ID NO:8), eEF2₈₁₉₋₈₂₇ (SEQID NO:9), eEF2₆₇₁₋₆₇₉ (SEQ ID NO:10), eEF2₆₆₁₋₆₆₉ (SEQ ID NO:11),eEF2₃₉₄₋₄₀₂ (SEQ ID NO:12) and eEF2₂₈₄₋₂₉₂ (SEQ ID NO:13)] bind to theHLA-A*0201 molecule and have a cytotoxic activity.

Next, evaluation was carried out as to whether the above 6 peptides andthe eEF2₂₉₂₋₃₀₀ peptide (SEQ ID NO:14) can bind to an HLA-A*0206molecule and produce interferon-γ. The experiments were carried out inthe same way as in the above method, except that donors having theHLA-A*0206 molecule were used. As a result, it was found that allpeptides tested increase the production of interferon-γ (FIG. 29).

Binding Affinity of Modified-type EEF2 Peptides to HLA-A*0201 Molecule

Next, a binding affinity of modified-type eEF2 peptides, in which anamino acid at position 2 and/or position 9 in the above 6 peptides(eEF2₇₃₉₋₇₄₇, eEF2₅₁₉₋₅₂₇, eEF2₆₇₁₋₆₇₈, eEF2₆₆₁₋₆₆₈, eEF2₃₈₄₋₄₀₂ andeEF2₂₈₄₋₂₉₂) as well as eEF2₂₉₂₋₃₀₀ peptide (SEQ ID NO:14) was alteredto another amino acid, was predicted using a program as described above(Tables 15 to 21).

TABLE 15Prediction of binding affinity of modified-type (SEQ ID NOs: 32 to 34) ofeEF2₇₃₉₋₇₄₇ peptide (SEQ ID NO: 8) to HLA-A*0201 molecule using two program(NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid Affinity BindingLog Real Log Peptide sequence (nM) level score score score eEF2₇₃₉₋₇₄₇RLMEPIYLV 3 SB 0.880 2426.739 7.7943 eEF2₇₃₉₋₇₄₇ 2M RMMEPIYLV 3 SB 0.8971752.645 7.4689 (SEQ ID NO: 32) eEF2₇₃₉₋₇₄₇ 9L RLMEPIYLL 4 SB 0.860745.355 6.6139 (SEQ ID NO: 33) eEF2₇₃₉₋₇₄₇ 2M9L RMMEPIYLL 3 SB 0.877538.312 6.2884 (SEQ ID NO: 34)

TABLE 16Prediction of binding affinity of modified-type (SEQ ID NOs: 35 to 37) ofeEF2₅₁₉₋₅₂₇ peptide (SEQ ID NO: 9) to HLA-A*0201 molecule using two program(NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid Affinity BindingLog Real Log Peptide sequence (nM) level score score score eEF2₅₁₉₋₅₂₇KLVEGLKRL 289 WB 0.476 705.066 6.5583 eEF2₅₁₉₋₅₂₇2M KMVEGLKRL 201 WB0.510 509.214 6.2329 (SEQ ID NO: 35) eEF2₅₁₉₋₅₂₇9V KLVEGLKRV 178 WB0.521 2295.564 7.7387 (SEQ ID NO: 36) eEF2₅₁₉₋₅₂₇2M9V KMVEGLKRV 112 WB0.563 1657.907 7.4133 SEQ ID NO: 37)

TABLE 17Prediction of binding affinity of modified-type (SEQ ID NOs: 38 to 40)of eEF2₆₇₁₋₆₇₉ peptide (SEQ ID NO: 10) to HLA-A*0201 molecule using twoprogram (NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid AffinityBinding Log Real Log Peptide sequence (nM) level score score scoreeEF2₆₇₁₋₆₇₉ YLNEIKDSV 11 SB 0.778 642.758 6.4658 eEF2₆₇₁₋₆₇₉2M YMNEIKDSV10 SB 0.780 464.214 6.1403 (SEQ ID NO: 38) eEF2₆₇₁₋₆₇₉9L YLNEIKDSL 20 SB0.723 197.418 5.2853 (SEQ ID NO: 39) eEF2₆₇₁₋₆₇₉2M9L YMNEIKDSL 21 SB0.718 142.580 4.9599 (SEQ ID NO: 40)

TABLE 18Prediction of binding affinity of modified-type (SEQ ID NOs: 41 to 43)of eEF2₆₆₁₋₆₆₉ peptide (SEQ ID NO: 11) to HLA-A*0201 molecule using twoprogram (NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid AffinityBinding Log Real Log Peptide sequence (nM) level score score scoreeEF2₆₆₁₋₆₆₉ ILTDITKGV 46 SB 0.644 484.777 6.1837 eEF2₆₆₁₋₆₆₉2M IMTDITKGV44 SB 0.649 350.117 5.8583 (SEQ ID NO: 41) eEF2₆₆₁₋₆₆₉9L ILTDITKGL 82 WB0.592 148.896 5.0032 (SEQ ID NO: 42) eEF2₆₆₁₋₆₆₉2M9L IMTDITKGL 88 WB0.585 107.536 4.6778 (SEQ ID NO: 43)

TABLE 19Prediction of binding affinity of modified-type (SEQ ID NOs: 44 to 46)of eEF2₃₉₄₋₄₀₂ peptide (SEQ ID NO: 12) to HLA-A*0201 molecule using twoprogram (NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid AffinityBinding Log Real Log Peptide sequence (nM) level score score scoreeEF2₃₉₄₋₄₀₂ LMMYISKMV 24 SB 0.704 315.959 5.7556 eEF2₃₉₄₋₄₀₂2L LLMYISKMV44 SB 0.648 437.482 6.0810 (SEQ ID NO: 44) eEF2₃₉₄₋₄₀₂9V LMMYISKML 83 WB0.591 97.045 4.5752 (SEQ ID NO: 45) eEF2₃₉₄₋₄₀₂2L9L LLMYISKML 139 WB0.544 134.369 4.9006 (SEQ ID NO: 46)

TABLE 20Prediction of binding affinity of modified-type (SEQ ID NOs: 47 to 49)of eEF2₂₈₄₋₂₉₂ peptide (SEQ ID NO: 13) to HLA-A*0201 molecule using twoprogram (NetMHC3.0 and ProPred) NetMHC3.0 ProPred Amino acid AffinityBinding Log Real Log Peptide sequence (nM) level score score scoreeEF2₂₈₄₋₂₉₂ KLPRTFCQL 1145 0.705 142.060 4.9562 eEF2₂₈₄₋₂₉₂M KMPRTFCQL983 0.363 102.599 4.6308 (SEQ ID NO: 47) eEF2₂₈₄₋₂₉₂9V KLPRTFCQV 331 WB0.463 462.521 6.1367 (SEQ ID NO: 48) eEF2₂₈₄₋₂₉₂2M9L KMPRTFCQV 228 WB0.498 334.043 5.8113 (SEQ ID NO: 49)

TABLE 21Prediction of binding affinity of modified-type (SEQ ID NOs: 15 to 17and 24) of eEF2₂₉₂₋₃₀₀ peptide (SEQ ID NO: 14) to HLA-A*0201 moleculeusing two program (NetMHC3.0 and ProPred) ProPred NetMHC3.0 Amino acidReal Log Affinity Binding Log Peptide sequence score score (nM) levelscore eEF2₂₉₂₋₃₀₀ LILDPIFKV 3290.05 8.10 8 SB 0.802 eEF2₂₉₂₋₃₀₀2LLLLDPIFKV 23927.65 10.08 3 SB 0.898 (SEQ ID NO :15) eEF2₂₉₂₋₃₀₀2MLMLDPIFKV 17281.08 9.76 3 SB 0.898 (SEQ ID NO: 16) eEF2₂₉₂₋₃₀₀2L9LLLLDPIFKL 7349.21 8.90 3 SB 0.872 (SEQ ID NO: 17) eEF2₂₉₂₋₃₀₀2M9LLMLDPIFKL 5307.76 8.58 3 SB 0.872 (SEQ ID NO: 18)

In addition, on modified-type peptides (SEQ ID NOs:15 to 17 and 24) ofeEF2₂₉₂₋₃₀₀ (SEQ ID NO:14) among the above peptides, a binding affinityto the HLA-A*0201 molecule (Table 22), cytotoxicity (FIGS. 22 to 25) andan interferon-γ activity (FIG. 26) were actually evaluated using amethod as described above.

TABLE 22 Binding assay (stabilization assay) ofmodified-type eEF2₂₉₂₋₃₀₀ peptides Amino acid Peptide sequence MIF% MIF increase Ns 4.96 Non-peptide 84.50 eEF2₂₉₂₋₃₀₀ LILDPIFKV 311.69268.86 eEF2₂₉₂₋₃₀₀ 2L LLLDPIFKV 338.80 300.95 eEF2₂₉₂₋₃₀₀ 2M LMLDPIFKV313.14 270.58 eEF2₂₉₂₋₃₀₀ 2L9L LLLDPIFKL 319.26 277.82 eEF2₂₉₂₋₃₀₀ 2M9LLMLDPIFKL 275.42 225.94

As a result, it was found that, among the modified-type eEF2₂₉₂₋₃₀₀peptides, eEF2₂₉₂₋₃₀₀ 2 L (a peptide having alteration of from I to L inan amino acid at position 2 in the eEF2₂₉₂₋₃₀₀ peptide, SEQ ID NO:15),eEF2₂₉₂₋₃₀₀ 2 M (a peptide having alteration of from I to M in an aminoacid at position 2 in the eEF2₂₉₂₋₃₀₀ peptide, SEQ ID NO:16),eEF2₂₉₂₋₃₀₀ 2L9L (a peptide having alteration of from I to L in an aminoacid at position 2 and of from V to L in an amino acid at position 9 inthe eEF2₂₉₂₋₃₀₀ peptide, SEQ ID NO:17) and eEF2₂₉₂₋₃₀₀ 2 M9L (a peptidehaving alteration of from I to M in an amino acid at position 2 and offrom V to L in an amino acid at position 9 in the eEF2₂₉₂₋₃₀₀ peptide,SEQ ID NO:24) have a binding affinity to the HLA-A*0201 molecule higherthan that of the original eEF2₂₉₂₋₃₀₀ peptide (Table 22), and increasecytotoxicity and interferon-γ activity in human (FIGS. 22 to 26, and28).

Example 5 Forced Expression of EEF2 in Cancer Cell Line

Cell clones were established in which an eEF2 expression vector or avacant expression vector was expressed in stomach cancer cell lineAZ-521. The eEF2 expression vector is one in which a nucleotide sequenceof an eEF2 gene is inserted into a restriction enzyme cleavage site:EcoRI of pcDNA3.1 (+) (Invitrogen). These cells were cultured withoutsynchronization, and doubling time of the cells was calculated from cellcounts after 48 and 72 hours from the beginning of culture. Furthermore,1×10⁵ cells were fixed with 80% ethanol and then allowed to stand in PBScontaining propidium iodide (PI, 5 μg/ml) and RNaseA (200 μg/ml) for 30minutes, and distribution of each phase of cell cycle was analyzed byflow cytometry (FIG. 13, left upper graph). Since the doubling time ofcells corresponds to length of the cell cycle, the time was thenmultiplied by proportional distribution of each phase of cell cycle tocalculate progression time of each phase (FIG. 13, right upper graph andlower table). As a result, it was observed that the cell count in a G2/Mphase decreases and the progression time is shortened in cells havingeEF2 expressed (FIG. 13). This suggests that the eEF2 acceleratesprogression of the G2/M phase.

Each of 5×10⁶ cells of stomach cancer cell line AZ-521 having eEF2forcibly expressed (2 clones) and AZ-521 having a control vacant vectorexpressed (2 clones) established as described above was mixed withMatrigel (Becton Dickinson), and the mixture was subcutaneously injectedinto left and right abdominal regions of nude mice to form a tumor. Thesize of the tumor was measured twice a week, and observed for 34 days.Volume (mm³) of the tumor was calculated by (minor axis)²×(majoraxis)³/2. The results of three experiments carried out separately oneach clone are shown (FIG. 14). From the results, it was shown that thevolume of the tumor remarkably increases in mice injected with cellshaving eEF2 forcibly expressed as compared with a control. FIG. 15 showsa typical example. The left tumor is caused by AZ-521 cells having avacant vector expressed, and the right tumor by AZ-521 cells having eEF2forcibly expressed.

Example 6 Identification of Novel Target Sequence of ShRNA Targeting atEEF2

In order to develop an shRNA (hereinafter, referred to as shEF2) whichcan efficiently inhibit expression of eEF2 by targeting at the eEF2 andinhibit growth of cancer, two sequences (hereinafter, referred to asshEF-1918 and shEF-2804) were newly selected which can be targets in aneEF2 sequence.

A target sequence at positions 1918-1947 (positions 1918-1947 from the5′ end of a DNA sequence encoding an eEF2 protein) in an eEF2 gene:5′-gcc tggccgagga catcgataaa ggcgagg-3′ (SEQ ID NO:18).

A target sequence at positions 2804-2833 (positions 2804-2833 from the5′ end of a DNA sequence encoding an eEF2 protein) in an eEF2 gene:5′-actcaac cataacactt gatgccgttt ctt-3′ (SEQ ID NO:19).

Construction of ShRNA

In order to construct an shRNA for the above sequences (SEQ ID NOs:18and 19), a DNA sequence [shEF-1918 or shEF-2804 (sense strand)]consisting of a sense sequence of a target sequence (30 bases)—a loopsequence (10 bases)—an antisense sequence (30 bases), and itscomplementary DNA sequence [shEF-1918 or shEF-2804 (antisense strand)]were chemically synthesized and then annealed, and the product wasinserted into SacI and KpnI recognition sites of tRNA-shRNA expressionvector, piGENE tRNA Pur (Clontech, Palo Alto, Calif.). Such DNAsequences inserted are shown below. In this connection, variations wereadded to a portion of a sense sequence of a target sequence so that anantisense strand is efficiently taken into RISC when an RNA having asequence transcribed is cleaved (shown by underlines in the followingsequences).

shEF-1918 (sense strand): (SEQ ID NO: 20)5′-(gcc tggccgagga catcgatgaa agcgtgg) cttcctgtca(cctcgcc tttatcgatg tcctcggcca ggc)-3′ shEF-1918 (antisense strand):(SEQ ID NO: 21) 3′-(cgg accggctcct gtagctactt tcgcacc) gaaggacagt(ggagcgg aaatagctac aggagccggt ccg)-5′ shEF-2804 (sense strand):(SEQ ID NO: 22) 5′-(actcaac cataacactt gataccattt gtt) cttcctgtca(aag aaacggcatc aagtgttatg gttgagt)-3′ shEF-2804 (antisense strand):(SEQ ID NO: 23) 3′-(tgagttg gtattgtgaa ctatggtaaa caa) gaaggacagt(ttc tttgccgtag ttcacaatac caactca)-5′

Cell Culture and Introduction of ShRNA

Lung cancer cell PC-14, pancreatic cancer cell PCI6, fibrosarcome cellHT-1080 and malignant glioma cell A172 were cultured in DMEM containing10% FBS. In order to introduce an shRNA, cells (1×10⁵) were washed twicewith PBS and then suspended in 250 μL of an FBS-free RPMI1640 medium,each 10 μg of shEF-1918, shEF-2804, or a shRNA vector for Luciferase,shLuc dissolved in 50 uL of an FBS-free RPMI1640 medium was added to thesuspension, and electroporation was carried out using Gene Pulsor II(BioRad) under a condition of 950 μFD and 175 V. Survival rate of cellswas about 90% under this condition. After the introduction of the shRNA,the number of living cells was counted, the cells were seeded at adensity of 1×10⁵ cells/mL, trypsin treatment was carried out after 72hours, and the number of cells was counted. As a result, shEF-1918 andshEF-2804 significantly inhibited cell proliferation in all four typesof cells analyzed as compared with shLuc (FIG. 27).

INDUSTRIAL APPLICABILITY

The present invention provides a method for detecting cancer using eEF2as a marker, a pharmaceutical composition for treatment or preventiontargeting at eEF2, an HLA-A*2402-restricted or HLA-A*0201-restrictedeEF2 peptide, a pharmaceutical composition containing them, and others,and is therefore applicable in the field of pharmaceuticals, forexample, in the field of development and production of preventive ortherapeutic pharmaceuticals for various hematopoietic organ tumors orsolid cancers highly expressing an eEF2 gene.

SEQUENCE LISTING FREE TEXT

SEQ ID NO:2: eEF2 siRNASEQ ID NO:3: eEF2 siRNASEQ ID NO:18: eEF2 1918-1947SEQ ID NO:19: eEF2 2804-2833SEQ ID NO:20: shEF-1918 senseSEQ ID NO:21: shEF-1918 antisenseSEQ ID NO:22: shEF-2804 senseSEQ ID NO:23: shEF-2804 antisenseSequence Listing

1. A method for the treatment or prevention of cancer in anHLA-A*2402-positive subject, comprising administering to the subject aneEF2 peptide having an amino acid sequence composed of contiguous aminoacids derived from an eEF2 protein, wherein the amino acid sequence isselected from the group consisting of: (a) Arg Phe Tyr Ala Phe Gly ArgVal Phe (SEQ ID NO:4); (b) Ala Phe Gly Arg Val Phe Ser Gly Leu (SEQ IDNO:5); (c) Arg Phe Asp Val His Asp Val Thr Leu (SEQ ID NO:6); (d) AlaTyr Leu Pro Val Asn Glu Ser Phe (SEQ ID NO:7); and (e) an amino acidsequence having substitution, deletion or addition of one or severalamino acids in the amino acid sequences as shown in (a) to (d).
 2. Themethod according to claim 1, wherein the amino acid sequence is Ala TyrLeu Pro Val Asn Glu Ser Phe (SEQ ID NO:7).
 3. The method according toany one of claims 1 to 2, wherein the cancer is selected from the groupconsisting of lung adenocarcinoma, small-cell lung cancer, esophagealcancer, stomach cancer, colon cancer, pancreatic duct cancer, malignantglioblastoma, malignant lymphoma and head-and-neck squamous cell cancer.